Methods of synthesizing substituted purine compounds

ABSTRACT

The present invention provides an efficient process for the synthesis of (2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol and hydrates thereof and methods for treating disorders in which DOT1-mediated protein methylation plays a part, such as cancer and neurological disorders, by administering these compounds and pharmaceutical compositions to subjects in need thereof. The present invention also provides novel crystalline forms of (2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol and hydrates thereof (Form A, Form B, and Form C), characterized by a unique X-ray diffraction pattern and Differential Scanning Calorimetry profile, as well as a unique crystalline structure.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/356,384 filed on Mar. 18, 2019 (now U.S. Pat. No. 10,968,247), whichis a continuation of U.S. application Ser. No. 15/635,032 filed on Jun.27, 2017, which is a continuation of U.S. application Ser. No.14/777,317 filed on Sep. 15, 2015 (now U.S. Pat. No. 9,738,679), whichis a U.S. National Phase application, filed under 35 U.S.C. § 371, ofInternational Application No. PCT/US2014/027481, filed Mar. 14, 2014,which claims priority to, and the benefit of, U.S. provisionalapplication No. 61/799,147, filed Mar. 15, 2013, the entire contents ofeach of which are incorporated herein by reference in their entireties.

BACKGROUND OF THE INVENTION

Disease-associated chromatin-modifying enzymes (e.g., DOT1L) play a rolein diseases such as proliferative disorders, metabolic disorders, andblood disorders. Thus, there is a need for the development of smallmolecules that are capable of modulating the activity of DOT1L.

SUMMARY OF THE INVENTION

The present invention is directed to2-(6-amino-9H-purin-9-yl)-5-(((3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol:

or a hydrate, salt, or crystalline form thereof.

The present invention is also directed to(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol:

or a hydrate, salt, or crystalline form thereof.

The present invention relates to a crystalline form of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol hydrate. The presentinvention relates to a crystalline form of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-dioltrihydrate.

The present invention relates to a crystalline form of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol hydrate (Form A)characterized by an X-ray powder diffraction (XRPD) pattern comprisingpeaks at about 5.5, 16.9, and 16.6° 2θ using Cu Kα radiation. In oneembodiment, the crystalline form (Form A) is characterized by an XRPDpattern comprising peaks at about 5.5, 16.9, 16.6, and 18.8° 2θ using CuKα radiation. In one embodiment, the crystalline form (Form A) ischaracterized by an XRPD pattern comprising peaks at about 5.5, 16.9,16.6, 18.8, 14.3, and 12.7° 2θ using Cu Kα radiation. In one embodiment,the crystalline form (Form A) is characterized by an XRPD patterncomprising peaks at about 5.5, 16.9, 16.6, 18.8, 14.3, 12.7, 21.8, 20.0,10.0, and 11.0° 2θ using Cu Kα radiation. In one embodiment, thecrystalline form (Form A) is characterized by an XRPD patternsubstantially similar to that set forth in FIG. 1 .

The present invention relates to a crystalline form of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol hydrate (Form A)characterized by a Differential Scanning Calorimetry (DSC) thermogramhaving a single maximum value at about 80.4° C.

The present invention relates to a crystalline form of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol hydrate (Form A)characterized by an XRPD pattern comprising peaks at about 5.5, 16.9,and 16.6° 2θ using Cu Kα radiation and by a DSC thermogram having asingle maximum value at about 80.4° C. In one embodiment, thecrystalline form (Form A) is characterized by an XRPD pattern comprisingpeaks at about 5.5, 16.9, 16.6, and 18.8° 2θ using Cu Kα radiation andby a DSC thermogram having a single maximum value at about 80.4° C. Inone embodiment, the crystalline form (Form A) is characterized by anXRPD pattern comprising peaks at about 5.5, 16.9, 16.6, 18.8, 14.3, and12.7° 2θ using Cu Kα radiation and by a DSC thermogram having a singlemaximum value at about 80.4° C. In one embodiment, the crystalline form(Form A) is characterized by an XRPD pattern comprising peaks at about5.5, 16.9, 16.6, 18.8, 14.3, 12.7, 21.8, 20.0, 10.0, and 11.0° 2θ usingCu Kα radiation and by a DSC thermogram having a single maximum value atabout 80.4° C. In one embodiment, the crystalline form (Form A) ischaracterized by an XRPD pattern substantially similar to that set forthin FIG. 1 and by a DSC thermogram having a single maximum value at about80.4° C.

The present invention relates to a crystalline form of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol hydrate (Form A)characterized by a DSC thermogram having two endotherms with onsets ofabout 39.3° C. and about 127.2° C.

The invention relates to a crystalline form of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diolhydrate (Form A) characterized by an XRPD pattern comprising peaks atabout 5.5, 16.9, and 16.6° 2θ using Cu Kα radiation and by a DSCthermogram having two endotherms with onsets of about 39.3° C. and about127.2° C. In one embodiment, the crystalline form (Form A) ischaracterized by an XRPD pattern comprising peaks at about 5.5, 16.9,16.6, and 18.8° 2θ using Cu Kα radiation and by a DSC thermogram havingtwo endotherms with onsets of about 39.3° C. and about 127.2° C. In oneembodiment, the crystalline form (Form A) is characterized by an XRPDpattern comprising peaks at about 5.5, 16.9, 16.6, 18.8, 14.3, and 12.7°2θ using Cu Kα radiation and by a DSC thermogram having two endothermswith onsets of about 39.3° C. and about 127.2° C. In one embodiment, thecrystalline form (Form A) is characterized by an XRPD pattern comprisingpeaks at about 5.5, 16.9, 16.6, 18.8, 14.3, 12.7, 21.8, 20.0, 10.0, and11.0° 2θ using Cu Kα radiation and by a DSC thermogram having twoendotherms with onsets of about 39.3° C. and about 127.2° C. In oneembodiment, the crystalline form (Form A) is characterized by an XRPDpattern substantially similar to that set forth in FIG. 1 and by a DSCthermogram having two endotherms with onsets of about 39.3° C. and about127.2° C.

The present invention relates to a crystalline form of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol trihydrate (Form B)characterized by an XRPD pattern comprising peaks at about 16.5, 20.5,and 5.2° 2θ using Cu Kα radiation. In one embodiment, the crystallineform (Form B) is characterized by an XRPD pattern comprising peaks atabout 16.5, 20.5, 5.2, and 14.2° 2θ using Cu Kα radiation. In oneembodiment, the crystalline form (Form B) is characterized by an XRPDpattern comprising peaks at about 16.5, 20.5, 5.2, 14.2, 18.0, and 10.4°2θ using Cu Kα radiation. In one embodiment, the crystalline form (FormB) is characterized by an XRPD pattern comprising peaks at about 16.5,20.5, 5.2, 14.2, 18.0, 10.4, 12.3, 10.0, 22.7, and 20.9° 2θ using Cu Kαradiation. In one embodiment, the crystalline form (Form B) ischaracterized by an XRPD pattern substantially similar to that set forthin FIG. 6 .

The present invention relates to a crystalline form of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol trihydrate (Form B)characterized by a DSC thermogram having a single maximum value at about132.3° C.

The present invention relates to a crystalline form of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol trihydrate (Form B)characterized by an XRPD pattern comprising peaks at about 16.5, 20.5,and 5.2° 2θ using Cu Kα radiation and by a DSC thermogram having asingle maximum value at about 132.3° C. In one embodiment, thecrystalline form (Form B) is characterized by an XRPD pattern comprisingpeaks at about 16.5, 20.5, 5.2, and 14.2° 2θ using Cu Kα radiation andby a DSC thermogram having a single maximum value at about 132.3° C. Inone embodiment, the crystalline form (Form B) is characterized by anXRPD pattern comprising peaks at about 16.5, 20.5, 5.2, 14.2, 18.0, and10.4° 2θ using Cu Kα radiation and by a DSC thermogram having a singlemaximum value at about 132.3° C. In one embodiment, the crystalline form(Form B) is characterized by an XRPD pattern comprising peaks at about16.5, 20.5, 5.2, 14.2, 18.0, 10.4, 12.3, 10.0, 22.7, and 20.9° 2θ usingCu Kα radiation and by DSC thermogram having a single maximum value atabout 132.3° C. In one embodiment, the crystalline form (Form B) ischaracterized by an XRPD pattern substantially similar to that set forthin FIG. 6 and further characterized by a DSC thermogram having a singlemaximum value at about 132.3° C.

The present invention relates to a crystalline form of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol trihydrate (Form B)characterized by a DSC thermogram having an endotherm with an onset ofabout 102.6° C.

The invention relates to a crystalline form of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol trihydrate (Form B) characterizedby an XRPD pattern comprising peaks at about 16.5, 20.5, and 5.2° 2θusing Cu Kα radiation and by a DSC thermogram having an endotherm withan onset of about 102.6° C. In one embodiment, the crystalline form(Form B) is characterized by an XRPD pattern comprising peaks at about16.5, 20.5, 5.2, and 14.2° 2θ using Cu Kα radiation and by a DSCthermogram having an endotherm with an onset of about 102.6° C. In oneembodiment, the crystalline form (Form B) is characterized by an XRPDpattern comprising peaks at about 16.5, 20.5, 5.2, 14.2, 18.0, and 10.4°2θ using Cu Kα radiation and by a DSC thermogram having an endothermwith an onset of about 102.6° C. In one embodiment, the crystalline form(Form B) is characterized by an XRPD pattern comprising peaks at about16.5, 20.5, 5.2, 14.2, 18.0, 10.4, 12.3, 10.0, 22.7, and 20.9° 2θ usingCu Kα radiation and by a DSC thermogram having an endotherm with anonset of about 102.6° C. In one embodiment, the crystalline form (FormB) is characterized by an XRPD pattern substantially similar to that setforth in FIG. 6 and by a DSC thermogram having an endotherm with anonset of about 102.6° C.

The present invention relates to a crystalline form of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol anhydrate (Form C)characterized by an XRPD pattern comprising peaks at about 16.9, 5.7,and 14.5° 2θ using Cu Kα radiation. In one embodiment, the crystallineform (Form C) is characterized by an XRPD pattern comprising peaks atabout 16.9, 5.7, 14.5, and 22.2° 2θ using Cu Kα radiation. In oneembodiment, the crystalline form (Form C) is characterized by an XRPDpattern comprising peaks at about 16.9, 5.7, 14.5, 22.2, 19.1, and 20.0°2θ using Cu Kα radiation. In one embodiment, the crystalline form (FormC) is characterized by an X-ray diffraction pattern comprising peaks atabout 16.9, 5.7, 14.5, 22.2, 19.1, 20.0, 11.3, 12.9, 10.0, and 23.7° 2θusing Cu Kα radiation. In one embodiment, the crystalline form (Form C)is characterized by an XRPD pattern substantially similar to that setforth in FIG. 11 .

The present invention relates to a crystalline form of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol anhydrate (Form C)characterized by a DSC thermogram having a single maximum value at about148.0° C.

The present invention relates to a crystalline form of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol anhydrate (Form C)characterized by an XRPD pattern comprising peaks at about 16.9, 5.7,and 14.5° 2θ using Cu Kα radiation and by a DSC thermogram having asingle maximum value at about 148.0° C. In one embodiment, thecrystalline form (Form C) is characterized by an XRPD pattern comprisingpeaks at about 16.9, 5.7, 14.5, and 22.2° 2θ using Cu Kα radiation andby a DSC thermogram having a single maximum value at about 148.0° C. Inone embodiment, the crystalline form (Form C) is characterized by anXRPD pattern comprising peaks at about 16.9, 5.7, 14.5, 22.2, 19.1, and20.0° 2θ using Cu Kα radiation and by a DSC thermogram having a singlemaximum value at about 148.0° C. In one embodiment, the crystalline form(Form C) is characterized by an XRPD pattern comprising peaks at about16.9, 5.7, 14.5, 22.2, 19.1, 20.0, 11.3, 12.9, 10.0, and 23.7° 2θ usingCu Kα radiation and by a DSC thermogram having a single maximum value atabout 148.0° C. In one embodiment, the crystalline form (Form C) ischaracterized by an XRPD pattern substantially similar to that set forthin FIG. 11 and by a DSC thermogram having a single maximum value atabout 148.0° C.

The present invention relates to a pharmaceutical composition comprisinga crystalline form of2-(6-amino-9H-purin-9-yl)-5-(((3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-dioland a pharmaceutically acceptable excipient or carrier. The presentinvention relates to a pharmaceutical composition comprising acrystalline form of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-dioland a pharmaceutically acceptable excipient or carrier. The presentinvention relates to a pharmaceutical composition comprising acrystalline form of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diolhydrate and a pharmaceutically acceptable excipient or carrier. Thepresent invention relates to a pharmaceutical composition comprising acrystalline form of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol trihydrate and apharmaceutically acceptable excipient or carrier. The present inventionrelates to a pharmaceutical composition comprising crystalline form of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol hydrate (Form A) and apharmaceutically acceptable excipient or carrier. The present inventionrelates to a pharmaceutical composition comprising a crystalline form of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol trihydrate(Form B) and a pharmaceutically acceptable excipient or carrier. Thepresent invention relates to a pharmaceutical composition comprising acrystalline form of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol anhydrate (Form C) anda pharmaceutically acceptable excipient or carrier.

The present invention relates to a process for preparing a crystallineform of2-(6-amino-9H-purin-9-yl)-5-(((3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol (e.g.,(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol) or a hydrate thereof, comprisingthe step of recrystallizing(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-(((3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diolin a solvent.

The present invention relates to a process for preparing a crystallineform of2-(6-amino-9H-purin-9-yl)-5-(((3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol (e.g.,(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol)or a hydrate thereof, by slow evaporation, solvent-mediated phasetransition, anti-solvent addition, solvent sweeping, or vapor diffusion.In one embodiment, the crystalline form of the present invention isprepared by slow evaporation, solvent-mediated phase transition, oranti-solvent addition

In one embodiment, the process comprises recrystallizing2-(6-amino-9H-purin-9-yl)-5-(((3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol in a mixture of acetonitrile andwater. In one embodiment, the process comprises recrystallizing2-(6-amino-9H-purin-9-yl)-5-(((3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol in a mixture of isopropyl alcoholand water. In one embodiment, the process comprises recrystallizing2-(6-amino-9H-purin-9-yl)-5-(((3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol in a mixture of acetonitrile andwater and then recrystallization in a mixture of isopropyl alcohol andwater. In one embodiment, the crystalline form is Form A, Form B, orForm C.

The present invention relates to a compound of formula I:

or a salt or solvate thereof, wherein:

R_(a), R_(b), R_(c), and R_(d) are each independently -M₂-T₂;

M₂ is a bond, S(O)₂, S(O), S, C(O), C(O)O, O, O—C₁-C₄ alkyl linker,C₁-C₄ alkyl linker, NH, or NR_(t);

R_(t) is C₁-C₆ alkyl;

T₂ is H, halogen, or R_(S4);

R_(S4) is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₈ cycloalkyl,C₆-C₁₀ aryl, 4 to 8-membered heterocycloalkyl, or 5 to 10-memberedheteroaryl;

R_(e) and R_(f) are each independently H or C₁-C₆ alkyl; and

x is 1, 2, 3, 4, 5, or 6,

wherein each of O—C₁-C₄ alkyl linker, C₁-C₄ alkyl linker, R_(t), andR_(S4) is optionally substituted with one or more substituents selectedfrom the group consisting of halogen, hydroxyl, carboxyl, cyano, C₁-C₆alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ alkoxyl, amino, mono-C₁-C₆alkylamino, di-C₁-C₆ alkylamino, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to6-membered heterocycloalkyl, and 5 to 6-membered heteroaryl.

The present invention relates to a process for preparing(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol or a salt or hydratethereof, comprising the steps of:

(1) reacting9-((3aR,4R,6R,6aR)-6-(aminomethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-purin-6-aminewith acetone to yield9-((3aR,4R,6R,6aR)-6-((isopropylamino)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-purin-6-amine;

(2) reacting9-((3aR,4R,6R,6aR)-6-((isopropylamino)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-purin-6-aminewith 3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutanoneto yield9-((3aR,4R,6R,6aR)-6-(((3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-purin-6-amine;and

(3) converting9-((3aR,4R,6R,6aR)-6-(((3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-purin-6-amineto(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-(((3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol.

The process of the present invention may further comprise step (4):recrystallizing(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-(((3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diolto yield(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol or a salt ofhydrate thereof.

The present invention relates to a process for preparing3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutanone or asalt thereof, comprising the steps of:

(1) converting pent-4-enoic acid to benzyl pent-4-enoate;

(2) converting benzyl pent-4-enoate to benzyl3-(2,2-dichloro-3-oxo-cyclobutyl)propanoate;

(3) converting benzyl 3-(2,2-dichloro-3-oxo-cyclobutyl)propanoate tobenzyl 3-(3-oxo-cyclobutyl)propanoate;

(4) converting benzyl 3-(3-oxo-cyclobutyl)propanoate to3-(3-oxo-cyclobutyl)propanoic acid;

(5) reacting 3-(3-oxocyclobutyl)propanoic acid with4-tert-butyl-2-nitroaniline to yieldN-(4-tert-butyl-2-nitrophenyl)-3-(3-oxo-cyclobutyl)propanamide; and

(6) convertingN-(4-tert-butyl-2-nitrophenyl)-3-(3-oxo-cyclobutyl)propanamide to3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutanone. Inone embodiment,3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutanone saltis a hydrochloride salt.

The present invention further relates to a process for preparing3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutanone or asalt thereof, comprising at least one step selected from:

(1) converting dicyclohexylammonium 3-(3-oxocyclobutyl)propanoate to3-(3-oxocyclobutyl)propanoyl chloride;

(2) reacting 3-(3-oxocyclobutyl)propanoyl chloride with4-tert-butyl-2-nitroaniline to yieldN-(4-tert-butyl-2-nitrophenyl)-3-(3-oxo-cyclobutyl)propanamide; and

(3) convertingN-(4-tert-butyl-2-nitrophenyl)-3-(3-oxo-cyclobutyl)propanamide to3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutanone.

The present invention relates to methods of treating or preventingcancer. The present invention provides methods of treating cancer. Thepresent invention also provides methods of preventing cancer. The methodincludes administering to a subject in need thereof a therapeuticallyeffective amount of2-(6-amino-9H-purin-9-yl)-5-(((3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol(e.g.,(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol)or a pharmaceutically acceptable salt or solvate thereof. The cancer canbe a hematological cancer. In one embodiment, the cancer is leukemia. Ina further embodiment, the cancer is acute myeloid leukemia, acutelymphocytic leukemia, or mixed lineage leukemia.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph indicating the XRPD of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diolhydrate (Form A).

FIG. 2 is a graph indicating the DSC curve of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diolhydrate (Form A).

FIG. 3 is a graph indicating the TGA curve of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diolhydrate (Form A).

FIG. 4 is a graph indicating the adsorption/desorption isotherm at 25°C. of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diolhydrate (Form A).

FIG. 5 is a graph indicating the XRPD overlay before and after DVSanalysis of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diolhydrate (Form A).

FIG. 6 is a graph indicating the XRPD overlay (top: experimental;bottom: simulated) of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-dioltrihydrate (Form B).

FIG. 7 is a graph indicating the DSC curve of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol trihydrate (Form B).

FIG. 8 is a graph indicating the TGA curve of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol trihydrate (Form B).

FIG. 9 is a graph indicating the water adsorption/desorption isotherm at25° C. of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-dioltrihydrate (Form B).

FIG. 10 is a graph indicating the XRPD overlay before and after DVSanalysis of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-dioltrihydrate (Form B).

FIG. 11 is a graph indicating the XRPD of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol anhydrate (Form C).

FIG. 12 is a graph indicating the DSC curve of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol anhydrate (Form C).

FIG. 13 is a graph indicating the TGA curve of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol anhydrate (Form C).

FIG. 14 is a graph indicating the VT-XRPD of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol trihydrate (Form B).

FIG. 15 is a graph indicating XRPD samples from slurry experiments usinga mixture of Form A and Form B at room temperature of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-dioltrihydrate.

FIG. 16 is a graph indicating XRPD samples from slurry experiments usinga mixture of Form A and Form B at 50° C. of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-dioltrihydrate.

FIG. 17 is a graph indicating XRPD samples from slurry experiments usinga mixture of Form B and Form C at room temperature of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diolanhydrate and trihydrate, respectively.

FIG. 18 is a graph indicating XRPD samples from slurry experiments ofForm A and Form B of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-dioltrihydrate in SGF at 37° C.

FIG. 19 is a graph indicating the XRPD overlay of Form A, Form B, andForm C of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diolhydrate, trihydrate, and anhydrate, respectively.

FIG. 20 is a picture indicating the morphology of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol trihydrate (Form B).

FIG. 21 is a graph indicating the XRPD of remaining solids in solubilitydetermination of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-dioltrihydrate (Form B).

FIG. 22 is a scheme indicating the pKa plot of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol.

FIG. 23 is a graph indicating the XRPD of samples from physicalstability of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-dioltrihydrate (Form B).

FIG. 24 is an XRPD of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-dioltrihydrate (Form B) from physical stability study.

FIG. 25 is an HPLC chromatogram of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diolhydrate and trihydrate (Form A and Form B, respectively).

FIG. 26 is a HPLC chromatogram of EP-1 trihydrate (x is 3) free base.

FIG. 27A is an image of EP-1 trihydrate (x is 3) at ×5 magnification.

FIG. 27B is an image of EP-1 trihydrate (x is 3) at ×20 magnification.

FIG. 28 is a ¹H NMR spectrum of EP-1 trihydrate (x is 3).

FIG. 29 is a variable temperature (VT) ¹H NMR spectrum of EP-1trihydrate (x is 3).

FIG. 30 is FTIR of EP-1 trihydrate (x is 3) free base.

FIG. 31 is a graph indicating the TGA and DSC of EP-1 trihydrate (x is3) free base.

FIG. 32 is a VT-XRPD diffractogram of EP-1 trihydrate (x is 3) freebase.

FIG. 33A is an image of EP-1 trihydrate (x is 3) free base at 25° C.taken during a VT-XRPD experiment.

FIG. 33B is an image of EP-1 trihydrate (x is 3) free base at 125° C.taken during a VT-XRPD experiment.

FIG. 33C is an image of EP-1 trihydrate (x is 3) free base at 150° C.taken during a VT-XRPD experiment.

FIG. 33D is an image of EP-1 trihydrate (x is 3) free base at roomtemperature taken during a VT-XRPD experiment.

FIG. 34 is a GVS kinetic plot of EP-1 trihydrate (x is 3) free base.

FIG. 35 is a GVS isotherm of EP-1 trihydrate (x is 3) free base.

FIG. 36 is an XRPD diffractogram of EP-1 trihydrate (x is 3) free basepost GVS.

FIG. 37 is a TGA plot of EP-1 trihydrate (x is 3) free base post-GVS.

FIG. 38 is a ¹H NMR spectrum of EP-1 trihydrate (x is 3) free basepost-GVS.

FIG. 39 is a ¹H NMR spectrum of EP-1 trihydrate (x is 3) free base afterheating EP-1 trihydrate (x is 3) free base at 180° C.

FIG. 40 is an overlay of the XRPD diffractograms of EP-1 trihydrate (xis 3) free base after solubility analysis.

FIG. 41 is an overlay of ¹H NMR spectra of EP-1 trihydrate (x is 3)residue at pH4 wet vs. at pH4 dry.

FIG. 42 is a HPLC chromatogram of EP-1 trihydrate (x is 3) residue at pH4 after a few days.

FIG. 43 is an overlay of XRPD diffractograms of EP-1 trihydrate (x is 3)free base at 40° C./75% RH after a week.

FIG. 44 is a HPLC chromatogram of EP-1 trihydrate (x is 3) free baseafter 24 h of UV light exposure.

FIG. 45 is a graph indicating the distribution of species from EP-1trihydrate (x is 3) free base with the pH calculated from theextrapolated aqueous result. pKa can be calculated from the intersectionof the different species (free base as triangles, mono-protonated formas squares, bis-protonated form as crosses and tri-protonated form asdiamonds).

FIG. 46 is a XRPD overlay of EP-1 trihydrate (x is 3) solids obtained inthe polymorphism assessment.

FIG. 47 is an overlay of ¹H NMR spectra of Form 1 (P1) and Form 2 (P2)of EP-1 trihydrate (x is 3) free base.

FIG. 48 is a XRPD overlay of EP-1 trihydrate (x is 3) solids from thesalt assessment.

FIG. 49 is TGA/DSC plot of EP-1 trihydrate (x is 3) obtained aftertreatment with benzene sulfonic acid in 1,4-dioxane.

FIG. 50 is a ¹H NMR spectrum of EP-1 trihydrate (x is 3) obtained aftertreatment with benzene sulfonic acid in IPA.

FIG. 51 is a XRPD overlay of EP-1 trihydrate (x is 3) solids obtainedfrom L-aspartic acid.

FIG. 52 is a ¹H NMR spectrum of EP-1 trihydrate (x is 3) obtained aftertreatment with L-aspartic acid.

FIG. 53 is a XRPD overlay of EP-1 trihydrate (x is 3) solids obtainedfrom L-glutamic acid.

FIG. 54 is a ¹H NMR spectrum of EP-1 trihydrate (x is 3) obtained aftertreatment with L-glutamic acid.

FIG. 55 is a XRPD overlay of EP-1 trihydrate (x is 3) solids obtainedfrom D-glucoheptonic acid.

FIG. 56 is a ¹H NMR spectrum of EP-1 trihydrate (x is 3) obtained aftertreatment with D-glucoheptonic acid.

FIG. 57 is a ¹H NMR spectrum of EP-1 trihydrate (x is 3) obtained aftertreatment with 1,5-naphthalene disulfonic acid.

FIG. 58 is a ¹H NMR spectrum of EP-1 trihydrate (x is 3) obtained aftertreatment with H₂SO₄.

FIG. 59 is a ¹H NMR spectrum of EP-1 trihydrate (x is 3) obtained aftertreatment with H₃PO₄.

FIG. 60 is a ¹H NMR spectrum of EP-1 trihydrate (x is 3) obtained aftertreatment with L-tartaric acid.

FIG. 61 is a ¹H NMR spectrum of EP-1 trihydrate (x is 3) obtained aftertreatment with citric acid.

FIG. 62 is a TGA/DSC plot of EP-1 trihydrate (x is 3) obtained aftertreatment with 1,5-naphthalene disulfonic acid.

FIG. 63 is a TGA/DSC plot of EP-1 trihydrate (x is 3) obtained aftertreatment with H₂SO₄.

FIG. 64 is a TGA/DSC plot of EP-1 trihydrate (x is 3) obtained aftertreatment with H₃PO₄.

FIG. 65 is a TGA/DSC plot of EP-1 trihydrate (x is 3) obtained aftertreatment with L-tartaric acid.

FIG. 66 is a TGA/DSC plot of EP-1 trihydrate (x is 3) obtained aftertreatment with citric acid.

FIG. 67 is a mDSC plot of EP-1 trihydrate (x is 3) obtained aftertreatment with H₂SO₄.

FIG. 68 is a mDSC plot of EP-1 trihydrate (x is 3) obtained aftertreatment with H₃PO₄.

FIG. 69 is a mDSC plot of EP-1 trihydrate (x is 3) obtained aftertreatment with L-tartaric acid.

FIG. 70 is a mDSC plot of EP-1 trihydrate (x is 3) obtained aftertreatment with citric acid.

FIG. 71 is a ¹H NMR spectrum of EP-1 trihydrate (x is 3) obtained aftertreatment with HCl.

FIG. 72 is a ¹H NMR spectrum of EP-1 trihydrate (x is 3) obtained aftertreatment with p-toluene sulfonic acid.

FIG. 73 is a ¹H NMR spectrum of EP-1 trihydrate (x is 3) obtained aftertreatment with methane sulfonic acid.

FIG. 74 is a ¹H NMR spectrum of EP-1 trihydrate (x is 3) obtained aftertreatment with maleic acid.

FIG. 75 is a ¹H NMR spectrum of EP-1 trihydrate (x is 3) obtained aftertreatment with adipic acid.

FIG. 76 is a ¹H NMR spectrum of EP-1 trihydrate (x is 3) obtained aftertreatment with succinic acid.

FIG. 77 is a ¹H NMR spectrum of EP-1 trihydrate (x is 3) obtained aftertreatment with malonic acid.

FIG. 78 is a ¹H NMR spectrum of EP-1 trihydrate (x is 3) obtained aftertreatment with fumaric acid.

FIG. 79 is a ¹H NMR spectrum of EP-1 trihydrate (x is 3) obtained aftertreatment with galactaric acid.

FIG. 80 is a ¹H NMR spectrum of EP-1 trihydrate (x is 3) obtained aftertreatment with D-glucoronic acid.

FIG. 81 is a ¹H NMR spectrum of EP-1 trihydrate (x is 3) obtained aftertreatment with lactobionic acid.

FIG. 82 is a ¹H NMR spectrum of EP-1 trihydrate (x is 3) obtained aftertreatment with malic acid.

FIG. 83 is a ¹H NMR spectrum of EP-1 trihydrate (x is 3) obtained aftertreatment with hippuric acid.

FIG. 84 is a ¹H NMR spectrum of EP-1 trihydrate (x is 3) obtained aftertreatment with gluconic acid.

FIG. 85 is a ¹H NMR spectrum of EP-1 trihydrate (x is 3) obtained aftertreatment with lactic acid.

FIG. 86 is a DSC plot of EP-1 trihydrate (x is 3) obtained aftertreatment with HCl.

FIG. 87 is a DSC plot of EP-1 trihydrate (x is 3) obtained aftertreatment with p-toluene sulfonic acid.

FIG. 88 is a DSC plot of EP-1 trihydrate (x is 3) obtained aftertreatment with methane sulfonic acid.

FIG. 89 is a DSC plot of EP-1 trihydrate (x is 3) obtained aftertreatment with benzene sulfonic acid.

FIG. 90 is a DSC plot of EP-1 trihydrate (x is 3) obtained aftertreatment with maleic acid.

FIG. 91 is a DSC plot of EP-1 trihydrate (x is 3) obtained aftertreatment with adipic acid.

FIG. 92 is a DSC plot of EP-1 trihydrate (x is 3) obtained aftertreatment with succinic acid.

FIG. 93 is a DSC plot of EP-1 trihydrate (x is 3) obtained aftertreatment with malonic acid.

FIG. 94 is a DSC plot of EP-1 trihydrate (x is 3) obtained aftertreatment with fumaric acid.

FIG. 95 is a DSC plot of EP-1 trihydrate (x is 3) obtained aftertreatment with galactaric acid.

FIG. 96 is a DSC plot of EP-1 trihydrate (x is 3) obtained aftertreatment with D-glucuronic acid.

FIG. 97 is a DSC plot of EP-1 trihydrate (x is 3) obtained aftertreatment with lactobionic acid.

FIG. 98 is a DSC plot of EP-1 trihydrate (x is 3) obtained aftertreatment with L-malic acid.

FIG. 99 is a DSC plot of EP-1 trihydrate (x is 3) obtained aftertreatment with hippuric acid.

FIG. 100 is a DSC plot of EP-1 trihydrate (x is 3) obtained aftertreatment with D-gluconic acid.

FIG. 101 is a DSC plot of EP-1 trihydrate (x is 3) obtained aftertreatment with lactic acid.

FIG. 102 is a mDSC plot of EP-1 trihydrate (x is 3) obtained aftertreatment with HCl.

FIG. 103 is a mDSC plot of EP-1 trihydrate (x is 3) obtained aftertreatment with p-toluene sulfonic acid.

FIG. 104 is a mDSC plot of EP-1 trihydrate (x is 3) obtained aftertreatment with methane sulfonic acid.

FIG. 105 is a mDSC plot of EP-1 trihydrate (x is 3) obtained aftertreatment with maleic acid.

FIG. 106 is a mDSC plot of EP-1 trihydrate (x is 3) obtained aftertreatment with adipic acid.

FIG. 107 is a mDSC plot of EP-1 trihydrate (x is 3) obtained aftertreatment with succinic acid.

FIG. 108 is a mDSC plot of EP-1 trihydrate (x is 3) obtained aftertreatment with malonic acid.

FIG. 109 is a mDSC plot of EP-1 trihydrate (x is 3) obtained aftertreatment with fumaric acid.

FIG. 110 is a mDSC plot of EP-1 trihydrate (x is 3) obtained aftertreatment with galactaric acid.

FIG. 111 is a mDSC plot of EP-1 trihydrate (x is 3) obtained aftertreatment with D-glucuronic acid.

FIG. 112 is a mDSC plot of EP-1 trihydrate (x is 3) obtained aftertreatment with L-malic acid.

FIG. 113 is a mDSC plot of EP-1 trihydrate (x is 3) obtained aftertreatment with gluconic acid.

FIG. 114 is a mDSC plot of EP-1 trihydrate (x is 3) obtained aftertreatment with lactic acid.

FIG. 115 is a ¹H NMR spectrum of EP-1 trihydrate (x is 3) H₂SO₄hemi-salt.

FIG. 116 is a ¹H NMR spectrum of EP-1 trihydrate (x is 3)1,5-naphthalene disulfonic hemi-salt.

FIG. 117 is a ¹H NMR spectrum of EP-1 trihydrate (x is 3) maleichemi-salt.

FIG. 118 is a ¹H NMR spectrum of EP-1 trihydrate (x is 3) H₃PO₄hemi-salt.

FIG. 119 is a ¹H NMR spectrum of EP-1 trihydrate (x is 3) HCl bis-salt.

FIG. 120 is a ¹H NMR spectrum of EP-1 trihydrate (x is 3)1,5-naphthalene disulfonic bis-salt.

FIG. 121 is a TGA/DSC plot of EP-1 trihydrate (x is 3) 1,5-naphthalenedisulfonic bis-salt.

FIG. 122 is a ¹H NMR spectrum of EP-1 trihydrate (x is 3) H₂SO₄bis-salt.

FIG. 123 is a TGA/DSC plot of EP-1 trihydrate (x is 3) H₂SO₄ bis-salt.

FIG. 124 is a ¹H NMR spectrum of EP-1 trihydrate (x is 3) p-toluenesulfonic bis-salt.

FIG. 125 is a ¹H NMR spectrum of EP-1 trihydrate (x is 3) methanesulfonic bis-salt.

FIG. 126 is a ¹H NMR spectrum of EP-1 trihydrate (x is 3) benzenesulfonic bis-salt.

FIG. 127 is a ¹H NMR spectrum of EP-1 trihydrate (x is 3) asparticbis-salt.

FIG. 128 is a ¹H NMR spectrum of EP-1 trihydrate (x is 3) maleicbis-salt.

FIG. 129 is a ¹H NMR spectrum of EP-1 trihydrate (x is 3) H₃PO₄bis-salt.

FIG. 130 is a TGA/DSC plot of EP-1 trihydrate (x is 3) H₃PO₄ bis-salt.

FIG. 131 is a ¹H NMR spectrum of EP-1 trihydrate (x is 3) salt obtainedby lyophilization from HCl.

FIG. 132 is a ¹H NMR spectrum of EP-1 trihydrate (x is 3) salt obtainedby lyophilization from p-toluene sulfonic acid.

FIG. 133 is a ¹H NMR spectrum of EP-1 trihydrate (x is 3) salt obtainedby lyophilization from methane sulfonic acid.

FIG. 134 is a ¹H NMR spectrum of EP-1 trihydrate (x is 3) salt obtainedby lyophilization from maleic acid.

FIG. 135 is a TGA/DSC plot of EP-1 trihydrate (x is 3) salt obtained bylyophilization from maleic acid.

FIG. 136 is a ¹H NMR spectrum of EP-1 trihydrate (x is 3) salt obtainedby lyophilization from adipic acid.

FIG. 137 is a ¹H NMR spectrum of EP-1 trihydrate (x is 3) salt obtainedby lyophilization from malonic acid.

FIG. 138 is a TGA/DSC plot of EP-1 trihydrate (x is 3) salt obtained bylyophilization from malonic acid.

FIG. 139 is a ¹H NMR spectrum of EP-1 trihydrate (x is 3) salt obtainedby lyophilization from fumaric acid.

FIG. 140 is a ¹H NMR spectrum of EP-1 trihydrate (x is 3) salt obtainedby lyophilization from lactobionic acid.

FIG. 141 is a ¹H NMR spectrum of EP-1 trihydrate (x is 3) salt obtainedby lyophilization from L-malic acid.

FIG. 142 is a TGA/DSC plot of EP-1 trihydrate (x is 3) salt obtained bylyophilization from L-malic acid.

FIG. 143 is a ¹H NMR spectrum of EP-1 trihydrate (x is 3) salt obtainedby lyophilization from hippuric acid.

FIG. 144 is a ¹H NMR spectrum of EP-1 trihydrate (x is 3) salt obtainedby lyophilization from D-gluconic acid.

FIG. 145A is an image of glass materials of EP-1 trihydrate (x is 3)observed under normal light.

FIG. 145B is an image of glass materials of EP-1 trihydrate (x is 3)observed under polarized light.

FIG. 146A is an image showing birefringence in a powder sample undernormal light.

FIG. 146B is an image showing birefringence in a powder sample underpolarized light.

FIG. 147 is a ¹H NMR spectrum of EP-1 trihydrate (x is 3)1,5-naphthalene disulfonic salt.

FIG. 148 is a TGA/DSC plot of EP-1 trihydrate (x is 3) 1,5-naphthalenedisulfonic salt.

FIG. 149 is a GVS kinetic plot of EP-1 trihydrate (x is 3)1,5-naphthalene disulfonic salt.

FIG. 150 is a GVS isotherm of EP-1 trihydrate (x is 3) 1,5-naphthalenedisulfonic salt.

FIG. 151 is a TGA plot after GVS of EP-1 trihydrate (x is 3)1,5-naphthalene disulfonic salt.

FIG. 152 is a mDSC plot of EP-1 trihydrate (x is 3) 1,5-naphthalenedisulfonic salt.

FIG. 153 is a ¹H NMR spectrum of EP-1 trihydrate (x is 3) H₂SO₄ salt.

FIG. 154 is a TGA/DSC plot of EP-1 trihydrate (x is 3) H₂SO₄ salt.

FIG. 155 is a mDSC plot of EP-1 trihydrate (x is 3) H₂SO₄ salt.

FIG. 156 is a ¹H NMR spectrum of EP-1 trihydrate (x is 3) H₃PO₄ salt.

FIG. 157 is a TGA/DSC plot of EP-1 trihydrate (x is 3) H₃PO₄ salt.

FIG. 158 is a mDSC plot of EP-1 trihydrate (x is 3) H₃PO₄ salt.

FIG. 159 is a ¹H NMR spectrum of EP-1 trihydrate (x is 3) H₃PO₄ salt.

FIG. 160 is a TGA/DSC plot of EP-1 trihydrate (x is 3) L-tartrate salt.

FIG. 161 is a mDSC plot of EP-1 trihydrate (x is 3) L-tartrate salt.

FIG. 162 is a GVS kinetic plot of EP-1 trihydrate (x is 3) L-tartratesalt.

FIG. 163 is a GVS isotherm of EP-1 trihydrate (x is 3) L-tartrate salt.

FIG. 164 is a ¹H NMR spectrum post-GVS of EP-1 trihydrate (x is 3)L-tartrate salt.

FIG. 165 is a ¹H NMR spectrum of EP-1 trihydrate (x is 3) citrate salt.

FIG. 166 is a TGA/DSC plot of EP-1 trihydrate (x is 3) citrate salt.

FIG. 167 is a mDSC plot of EP-1 trihydrate (x is 3) citrate salt.

FIG. 168 is a GVS kinetic plot of EP-1 trihydrate (x is 3) citrate salt.

FIG. 169 is a GVS isotherm of EP-1 trihydrate (x is 3) citrate salt.

FIG. 170 is a DSC plot post-GVS of EP-1 trihydrate (x is 3) citratesalt.

FIG. 171 is a ¹H NMR spectrum post-GVS of EP-1 trihydrate (x is 3)citrate salt.

FIG. 172 is a GVS kinetic plot at 70% RH of EP-1 trihydrate (x is 3)citrate salt.

FIG. 173 is a TGA/DSC post-GVS at 70% RH of EP-1 trihydrate (x is 3)citrate salt.

FIG. 174 is a ¹H NMR spectrum of EP-1 trihydrate (x is 3) p-toluenesulfonate salt.

FIG. 175 is a TGA/DSC plot of EP-1 trihydrate (x is 3) p-toluenesulfonate salt.

FIG. 176 is a mDSC plot of EP-1 trihydrate (x is 3) p-toluene sulfonatesalt.

FIG. 177 is a ¹H NMR spectrum of EP-1 trihydrate (x is 3) methanesulfonate salt.

FIG. 178 is a TGA/DSC plot of EP-1 trihydrate (x is 3) methane sulfonatesalt.

FIG. 179 is a mDSC plot of EP-1 trihydrate (x is 3) methane sulfonatesalt.

FIG. 180 is a ¹H NMR spectrum of EP-1 trihydrate (x is 3) HCl salt.

FIG. 181 is a TGA/DSC plot of EP-1 trihydrate (x is 3) HCl salt.

FIG. 182 is a mDSC plot of EP-1 trihydrate (x is 3) HCl salt.

FIG. 183 is a GVS kinetic plot of EP-1 trihydrate (x is 3) HCl salt.

FIG. 184 is a GVS isotherm of EP-1 trihydrate (x is 3) HCl salt.

FIG. 185 is a ¹H NMR spectrum post-GVS of EP-1 trihydrate (x is 3) HClsalt.

FIG. 186 is a DSC plot post-GVS of EP-1 trihydrate (x is 3) HCl salt.

FIG. 187 is an overlap of XRPD diffractograms for EP-1 trihydrate (x is3) free base (Form 1 &2) and HCl salt after 3 weeks at 25° C./75% RH121.

FIG. 188 is a ¹H NMR spectrum of EP-1 trihydrate (x is 3) HCl salt after3 weeks at 25° C./75% RH.

FIG. 189A is an image of EP-1 trihydrate (x is 3) HCl salt after 3 weeksat 25° C./75% RH under normal light.

FIG. 189B is an image of EP-1 trihydrate (x is 3) HCl salt after 3 weeksat 25° C./75% RH under polarized light.

FIG. 190 is a TGA overlay of EP-1 trihydrate (x is 3) free base, EP-1trihydrate (x is 3) HCl salt, and EP-1 trihydrate (x is 3) HCl saltafter 3 weeks at 25° C./75% RH.

FIG. 191 is a DSC overlay of EP-1 trihydrate (x is 3) free base, EP-1trihydrate (x is 3) HCl salt, and EP-1 trihydrate (x is 3) HCl saltafter 3 weeks at 25° C./75% RH.

FIG. 192 is a ¹H NMR spectrum of compound 10B as described in Example2A.

FIG. 193 is a ¹H NMR spectrum of compound 12 as described in Example 2A.

FIG. 194 is a HPLC chromatogram of compound 3 as described in Example2A.

FIG. 195 is a ¹H NMR spectrum of compound 3 as described in Example 2A.

FIG. 196 is a LC-MS spectrum of compound 3 as described in Example 2A.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to2-(6-amino-9H-purin-9-yl)-5-(((3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol:

or a hydrate, salt, or crystalline form thereof.

The present invention is also directed to(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol:

or a hydrate, salt, or crystalline form thereof.

The present invention is also directed to a crystalline form of2-(6-amino-9H-purin-9-yl)-5-(((3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diolor a hydrate or salt thereof. The present invention is also directed toa crystalline form of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol or a hydrate or salt thereof.

In one embodiment, the crystalline form of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol is Form A, characterized by anXRPD pattern comprising peaks at about 5.5, 16.9, and 16.6° 2θ using CuKα radiation. In one embodiment, Form A is characterized by an XRPDpattern comprising peaks at about 5.5, 16.9, 16.6, and 18.8° 2θ using CuKα radiation. In one embodiment, Form A is characterized by an XRPDpattern comprising peaks at about 5.5, 16.9, 16.6, 18.8, 14.3, and 12.7°2θ using Cu Kα radiation. In one embodiment, Form A is characterized byan XRPD pattern comprising peaks at about 5.5, 16.9, 16.6, 18.8, 14.3,12.7, 21.8, 20.0, 10.0, and 11.0° 2θ using Cu Kα radiation. In oneembodiment, Form A is characterized by an XRPD pattern substantiallysimilar to that set forth in FIG. 1 .

In one embodiment, Form A is characterized by a DSC thermogram having asingle maximum value at about 80.4° C. In one embodiment, Form A ischaracterized by a DSC thermogram having two endotherms with onsets ofabout 39.3° C. and about 127.2° C.

In one embodiment, Form A is characterized by an XRPD pattern comprisingpeaks at about 5.5, 16.9, and 16.6° 2θ using Cu Kα radiation and by aDSC thermogram having a single maximum value at about 80.4° C. or by aDSC thermogram having two endotherms with onsets of about 39.3° C. andabout 127.2° C. In one embodiment, Form A is characterized by an XRPDpattern comprising peaks at about 5.5, 16.9, 16.6, and 18.8° 2θ using CuKα radiation and by a DSC thermogram having a single maximum value atabout 80.4° C. or by a DSC thermogram having two endotherms with onsetsof about 39.3° C. and about 127.2° C. In one embodiment, Form A ischaracterized by an XRPD pattern comprising peaks at about 5.5, 16.9,16.6, 18.8, 14.3, and 12.7° 2θ using Cu Kα radiation and by a DSCthermogram having a single maximum value at about 80.4° C. or by a DSCthermogram having two endotherms with onsets of about 39.3° C. and about127.2° C. In one embodiment, Form A is characterized by an XRPD patterncomprising peaks at about 5.5, 16.9, 16.6, 18.8, 14.3, 12.7, 21.8, 20.0,10.0, and 11.0° 2θ using Cu Kα radiation and by a DSC thermogram havinga single maximum value at about 80.4° C. or by a DSC thermogram havingtwo endotherms with onsets of about 39.3° C. and about 127.2° C. In oneembodiment, Form A is characterized by an XRPD pattern substantiallysimilar to that set forth in FIG. 1 and by a DSC thermogram having asingle maximum value at about 80.4° C. or by a DSC thermogram having twoendotherms with onsets of about 39.3° C. and about 127.2° C.

In one embodiment, the crystalline form of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol is characterized by an XRPDpattern comprising at least three peaks selected from the groupconsisting of about 5.5, 16.9, 16.6, 18.8, 14.3, 12.7, 21.8, 20.0, 10.0,and 11.0° 2θ using Cu Kα radiation, by an XRPD pattern comprising atleast four peaks selected from the group consisting of about 5.5, 16.9,16.6, 18.8, 14.3, 12.7, 21.8, 20.0, 10.0, and 11.0° 2θ using Cu Kαradiation, by an XRPD pattern comprising at least five peaks selectedfrom the group consisting of about 5.5, 16.9, 16.6, 18.8, 14.3, 12.7,21.8, 20.0, 10.0, and 11.0° 2θ using Cu Kα radiation, by an XRPD patterncomprising at least six peaks selected from the group consisting ofabout 5.5, 16.9, 16.6, 18.8, 14.3, 12.7, 21.8, 20.0, 10.0, and 11.0° 2θusing Cu Kα radiation, by an XRPD pattern comprising at least sevenpeaks selected from the group consisting of about 5.5, 16.9, 16.6, 18.8,14.3, 12.7, 21.8, 20.0, 10.0, and 11.0° 2θ using Cu Kα radiation, or byan XRPD pattern comprising at least eight peaks selected from the groupconsisting of about 5.5, 16.9, 16.6, 18.8, 14.3, 12.7, 21.8, 20.0, 10.0,and 11.0° 2θ using Cu Kα radiation.

In one embodiment, the crystalline form of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol is Form A, characterized as shownin Table A below.

TABLE A Selected XRPD diffraction peaks of Form A Angle 2- Theta (2θ)d-spacing (Å) Intensity % 5.5 15.9835 100 16.9 5.2468 59.49 16.6 5.355845.67 18.8 4.7245 38.23 14.3 6.1836 34.91 12.7 6.9600 30.34 21.8 4.075624.26 20.0 4.4336 21.36 10.0 8.8499 17.84 11.0 8.0267 17.29

In one embodiment, the crystalline form of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diolis Form B, characterized by an XRPD pattern comprising peaks at about16.5, 20.5, and 5.2° 2θ using Cu Kα radiation. In one embodiment, thecrystalline form (Form B) is characterized by an XRPD pattern comprisingpeaks at about 16.5, 20.5, 5.2, and 14.2° 2θ using Cu Kα radiation. Inone embodiment, the crystalline form (Form B) is characterized by anXRPD pattern comprising peaks at about 16.5, 20.5, 5.2, 14.2, 18.0, and10.4° 2θ using Cu Kα radiation. In one embodiment, the crystalline form(Form B) is characterized by an XRPD pattern comprising peaks at about16.5, 20.5, 5.2, 14.2, 18.0, 10.4, 12.3, 10.0, 22.7, and 20.9° 2θ usingCu Kα radiation. In one embodiment, the crystalline form (Form B) ischaracterized by an XRPD pattern substantially similar to that set forthin FIG. 6 .

In one embodiment, Form B is characterized by a DSC thermogram having asingle maximum value at about 132.3° C. In one embodiment, Form B ischaracterized by a DSC thermogram having an endotherm with an onset ofabout 102.6° C.

In one embodiment, Form B is characterized by an XRPD pattern comprisingpeaks at about 16.5, 20.5, and 5.2° 2θ using Cu Kα radiation and by aDSC thermogram having a single maximum value at about 132.3° C. or by aDSC thermogram having an endotherm with an onset of about 102.6° C. Inone embodiment, the crystalline form (Form B) is characterized by anXRPD pattern comprising peaks at about 16.5, 20.5, 5.2, and 14.2° 2θusing Cu Kα radiation and by a DSC thermogram having a single maximumvalue at about 132.3° C. or by a DSC thermogram having an endotherm withan onset of about 102.6° C. In one embodiment, the crystalline form(Form B) is characterized by an XRPD pattern comprising peaks at about16.5, 20.5, 5.2, 14.2, 18.0, and 10.4° 2θ using Cu Kα radiation and by aDSC thermogram having a single maximum value at about 132.3° C. or by aDSC thermogram having an endotherm with an onset of about 102.6° C. Inone embodiment, the crystalline form (Form B) is characterized by anXRPD pattern comprising peaks at about 16.5, 20.5, 5.2, 14.2, 18.0,10.4, 12.3, 10.0, 22.7, and 20.9° 2θ using Cu Kα radiation and by a DSCthermogram having a single maximum value at about 132.3° C. or by a DSCthermogram having an endotherm with an onset of about 102.6° C. In oneembodiment, the crystalline form (Form B) is characterized by an XRPDpattern substantially similar to that set forth in FIG. 6 and by a DSCthermogram having a single maximum value at about 132.3° C. or by a DSCthermogram having an endotherm with an onset of about 102.6° C.

In one embodiment, the crystalline form of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol is characterized by an XRPDpattern comprising at least three peaks selected from the groupconsisting of about 16.5, 20.5, 5.2, 14.2, 18.0, 10.4, 12.3, 10.0, 22.7,and 20.9° 2θ using Cu Kα radiation, by an XRPD pattern comprising atleast four peaks selected from the group consisting of about 16.5, 20.5,5.2, 14.2, 18.0, 10.4, 12.3, 10.0, 22.7, and 20.9° 2θ using Cu Kαradiation, by an XRPD pattern comprising at least five peaks selectedfrom the group consisting of about 16.5, 20.5, 5.2, 14.2, 18.0, 10.4,12.3, 10.0, 22.7, and 20.9° 2θ using Cu Kα radiation, by an XRPD patterncomprising at least six peaks selected from the group consisting ofabout 16.5, 20.5, 5.2, 14.2, 18.0, 10.4, 12.3, 10.0, 22.7, and 20.9° 2θusing Cu Kα radiation, by an XRPD pattern comprising at least sevenpeaks selected from the group consisting of about 16.5, 20.5, 5.2, 14.2,18.0, 10.4, 12.3, 10.0, 22.7, and 20.9° 2θ using Cu Kα radiation, or byan XRPD pattern comprising at least eight peaks selected from the groupconsisting of about 16.5, 20.5, 5.2, 14.2, 18.0, 10.4, 12.3, 10.0, 22.7,and 20.9° 2θ using Cu Kα radiation.

In one embodiment, the crystalline form of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol is Form B, characterized as shownin Table B below.

TABLE B Selected XRPD diffraction peaks of Form B Angle 2- Theta (2θ)d-spacing (Å) Intensity % 16.5 5.3655 100 20.5 4.3247 99.11 5.2 16.976489.5 14.2 6.2371 86.31 18.0 4.9360 79.87 10.4 8.5065 56.50 12.3 7.179952.56 10.0 8.8652 40.06 22.7 3.9245 38.12 20.9 4.2505 34.10

In one embodiment, the crystalline form of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diolis Form C, characterized by an XRPD pattern comprising peaks at about16.9, 5.7, and 14.5° 2θ using Cu Kα radiation. In one embodiment, thecrystalline form (Form C) is characterized by an XRPD pattern comprisingpeaks at about 16.9, 5.7, 14.5, and 22.2° 2θ using Cu Kα radiation. Inone embodiment, the crystalline form (Form C) is characterized by anXRPD pattern comprising peaks at about 16.9, 5.7, 14.5, 22.2, 19.1, and20.0° 2θ using Cu Kα radiation. In one embodiment, the crystalline form(Form C) is characterized by an X-ray diffraction pattern comprisingpeaks at about 16.9, 5.7, 14.5, 22.2, 19.1, 20.0, 11.3, 12.9, 10.0, and23.7° 2θ using Cu Kα radiation. In one embodiment, the crystalline form(Form C) is characterized by an XRPD pattern substantially similar tothat set forth in FIG. 11 .

In one embodiment, Form C is characterized by a DSC thermogram having asingle maximum value at about 148.0° C.

In one embodiment, Form C is characterized by an XRPD pattern comprisingpeaks at about 16.9, 5.7, and 14.5° 2θ using Cu Kα radiation and by aDSC thermogram having a single maximum value at about 148.0° C. In oneembodiment, the crystalline form (Form C) is characterized by an XRPDpattern comprising peaks at about 16.9, 5.7, 14.5, and 22.2° 2θ using CuKα radiation and by a DSC thermogram having a single maximum value atabout 148.0° C. In one embodiment, the crystalline form (Form C) ischaracterized by an XRPD pattern comprising peaks at about 16.9, 5.7,14.5, 22.2, 19.1, and 20.0° 2θ using Cu Kα radiation and by a DSCthermogram having a single maximum value at about 148.0° C. In oneembodiment, the crystalline form (Form C) is characterized by an X-raydiffraction pattern comprising peaks at about 16.9, 5.7, 14.5, 22.2,19.1, 20.0, 11.3, 12.9, 10.0, and 23.7° 2θ using Cu Kα radiation and bya DSC thermogram having a single maximum value at about 148.0° C. In oneembodiment, the crystalline form (Form C) is characterized by an XRPDpattern substantially similar to that set forth in FIG. 11 and by a DSCthermogram having a single maximum value at about 148.0.

In one embodiment, the crystalline form of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol is characterized by an XRPDpattern comprising at least three peaks selected from the groupconsisting of about 16.9, 5.7, 14.5, 22.2, 19.1, 20.0, 11.3, 12.9, 10.0,and 23.7° 2θ using Cu Kα radiation, by an XRPD pattern comprising atleast four peaks selected from the group consisting of about 16.9, 5.7,14.5, 22.2, 19.1, 20.0, 11.3, 12.9, 10.0, and 23.7° 2θ using Cu Kαradiation, by an XRPD pattern comprising at least five peaks selectedfrom the group consisting of about 16.9, 5.7, 14.5, 22.2, 19.1, 20.0,11.3, 12.9, 10.0, and 23.7° 2θ using Cu Kα radiation, by an XRPD patterncomprising at least six peaks selected from the group consisting ofabout 16.9, 5.7, 14.5, 22.2, 19.1, 20.0, 11.3, 12.9, 10.0, and 23.7° 2θusing Cu Kα radiation, by an XRPD pattern comprising at least sevenpeaks selected from the group consisting of about 16.9, 5.7, 14.5, 22.2,19.1, 20.0, 11.3, 12.9, 10.0, and 23.7° 2θ using Cu Kα radiation, or byan XRPD pattern comprising at least eight peaks selected from the groupconsisting of about 16.9, 5.7, 14.5, 22.2, 19.1, 20.0, 11.3, 12.9, 10.0,and 23.7° 2θ using Cu Kα radiation.

In one embodiment, the crystalline form of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol is Form C, characterized as shownin Table C below.

TABLE C Selected XRPD diffraction peaks of Form C Angle 2- Theta (2θ)d-spacing (Å) Intensity % 16.9 5.2410 100 5.7 15.6364 88.18 14.5 6.114444.82 22.2 3.9984 39.09 19.1 4.6404 31.18 20.0 4.4421 30.79 11.3 7.820425.55 12.9 6.8744 22.74 10.0 8.8715 17.84 23.7 3.7529 10.23

When it is stated that the present invention relates to a crystallineform of Form A, Form B, or Form C, the degree of crystallinity isconveniently greater than about 60%, more conveniently greater thanabout 80%, preferably greater than about 90% and more preferably greaterthan about 95%. Most preferably the degree of crystallinity is greaterthan about 98%.

It will be understood that the 2-theta values of the XRPD pattern mayvary slightly from one machine to another or from one sample to another,and so the values quoted are not to be construed as absolute.

It is known that an XRPD pattern may be obtained which has one or moremeasurement errors depending on measurement conditions (such asequipment or machine used). In particular, it is generally known thatintensities in an XRPD pattern may fluctuate depending on measurementconditions. Therefore it should be understood that the Form A, Form B,and Form C of the present invention are not limited to the crystals thatprovide XRPD patterns identical to the XRPD pattern shown in Figures ofthe present invention and any crystals providing XRPD patternssubstantially the same as those shown in Figures of the presentinvention fall within the scope of the present invention. A personskilled in the art of XRPD is able to judge the substantial identity ofXRPD patterns.

Persons skilled in the art of XRPD will realize that the relativeintensity of peaks can be affected by, for example, grains above 30microns in size and non-unitary aspect ratios, which may affect analysisof samples. The skilled person will also realize that the position ofreflections can be affected by the precise height at which the samplesits in the diffractometer and the zero calibration of thediffractometer. The surface planarity of the sample may also have asmall effect. Hence the diffraction pattern data presented are not to betaken as absolute values. (Jenkins, R & Snyder, R. L. ‘Introduction toX-Ray Powder Diffractometry’ John Wiley & Sons 1996; Bunn, C. W. (1948),Chemical Crystallography, Clarendon Press, London; Klug, H. P. &Alexander, L. E. (1974), X-Ray Diffraction Procedures).

Generally, a measurement error of a diffraction angle in an X-ray powderdiffractogram is approximately plus or minus 0.2° 2-theta, and suchdegree of a measurement error should be taken into account whenconsidering the XRPD patterns presented in the Figures and Tables of thepresent invention. Furthermore, it should be understood that intensitiesmight fluctuate depending on experimental conditions and samplepreparation (preferred orientation). Any crystal form that provides aXRPD diffractogram, Raman/IR spectrum, SSNMR spectrum or DSC thermogramsubstantially identical to those disclosed herein, fall within the scopeof the present disclosures. One skilled in the art will have the abilityto determine substantial identities of diffractograms, spectra andthermograms.

In another aspect, the present invention is directed to a process forpreparing a crystalline form of2-(6-amino-9H-purin-9-yl)-5-(((3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol(e.g.,(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol)or a hydrate thereof. In one embodiment, the crystalline forms of thepresent invention can be prepared by slow evaporation, solvent-mediatedphase transition, anti-solvent addition, solvent sweeping, or vapordiffusion. In one embodiment, the crystalline form of the presentinvention can be prepared by slow evaporation, solvent-mediated phasetransition, or anti-solvent addition.

In one embodiment, the crystalline form of the present invention isprepared by slow evaporation. In one embodiment,2-(6-amino-9H-purin-9-yl)-5-(((3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diolis dissolved in a solvent. In one embodiment, the solvent is water, analkane, a haloalkane, a nitrile, an alcohol, a carboxylic acid, anester, an ether, toluene, tetrahydrofuran (THF), an acetone, a glycol,or a combination thereof. In one embodiment, the solvent isbenzonitrile, acetonitrile, THF, ethyl acetate, dichloroethane, toluene,isopropyl acetate, isopropyl alcohol, cylcohexanol, ethyl alcohol,methyl t-butyl ether (MTBE), water, acetone, glycol, hexane, or acombination thereof.

In one embodiment, after2-(6-amino-9H-purin-9-yl)-5-(((3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diolis dissolved in the solvent, the solvent is evaporated slowly toprecipitate2-(6-amino-9H-purin-9-yl)-5-(((3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol.In one embodiment, the solvent is evaporated over a period of 6 hours,12 hours, 18 hours, 24 hours, 48 hours, 72 hours, 96 hours, or 120hours, or more. In one embodiment, the solvent is evaporated over aperiod sufficient to allow2-(6-amino-9H-purin-9-yl)-5-(((3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol to precipitate from the solvent.In one embodiment, the solvent is evaporated over a period until thesolvent completely disappears.

In one embodiment, the slow evaporation is conducted under ambienttemperature (e.g., room temperature). In one embodiment, the slowevaporation is conducted at an elevated temperature (e.g., 20° C., 25°C., 30° C., 35° C., 40° C., 45° C., 50° C., 55° C., or 60° C., orhigher). In one embodiment, the slow evaporation is conducted at normalpressure (e.g., atmospheric pressure). In one embodiment, the slowevaporation is conducted at a reduced pressure (e.g., 0.9, 0.8, 0.7, 06,0.5, 0.4, 0.3, 0.2, or 0.1 of the atmospheric pressure, or lower).

In one embodiment, the crystalline form of the present invention isprepared by solvent-mediated phase transition. In one embodiment,2-(6-amino-9H-purin-9-yl)-5-(((3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diolis dissolved or suspended in a solvent. In one embodiment, the solventis water, an alkane, a haloalkane, an oxane, a nitrile, an alcohol, anester, an ether, toluene, THF, an acetone, or a combination thereof. Inone embodiment, the solvent is acetonitrile, THF, methyl THF,trifluoromethane, dioxane, heptane, toluene, isopropyl acetate,isopropyl alcohol, ethyl alcohol, methanol, MTBE, water, acetone, or acombination thereof.

In one embodiment, the solvent-mediated phase transition is conductedover a period of 6 hours, 12 hours, 18 hours, 24 hours, 48 hours, 72hours, 96 hours, or 120 hours or more.

In one embodiment, the solvent-mediated phase transition is conductedunder ambient temperature (e.g., room temperature). In one embodiment,the solvent-mediated phase transition is conducted at an elevatedtemperature (e.g., 20° C., 25° C., 30° C., 35° C., 40° C., 45° C., 50°C., 55° C., or 60° C., or higher). In one embodiment, thesolvent-mediated phase transition is conducted at normal pressure (e.g.,atmospheric pressure). In one embodiment, the solvent-mediated phasetransition is conducted at a reduced pressure (e.g., 0.9, 0.8, 0.7, 06,0.5, 0.4, 0.3, 0.2, or 0.1 of the atmospheric pressure, or lower). Inone embodiment, the solution or suspension is stirred.

In one embodiment, the crystalline form of the present invention isprepared by anti-solvent addition. In one embodiment,2-(6-amino-9H-purin-9-yl)-5-(((3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diolis dissolved in a solvent. In one embodiment, the solvent is an oxane, anitrile, an alcohol, THF, an acetone, a carboxylic acid, or acombination thereof. In one embodiment, the solvent is acetonitrile,THF, dioxane, isopropyl alcohol, ethyl alcohol, methanol, acetone,acetic acid, or a combination thereof. In one embodiment,2-(6-amino-9H-purin-9-yl)-5-(((3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diolis saturated in the solvent.

In one embodiment, an anti-solvent is added to the solution of2-(6-amino-9H-purin-9-yl)-5-(((3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol.In one embodiment, the anti-solvent is an alkane, water, or acombination thereof. In one embodiment, the anti-solvent is hexane orwater.

In one embodiment, the anti-solvent addition is conducted over a periodof 6 hours, 12 hours, 18 hours, 24 hours, 48 hours, 72 hours, 96 hours,or 120 hours or 6 days, 7 days, 10 days, 14 days, or more. In oneembodiment, the anti-solvent addition is conducted over a periodsufficient to allow2-(6-amino-9H-purin-9-yl)-5-(((3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diolto precipitate from the solvent.

In one embodiment, the anti-solvent addition is conducted under ambienttemperature (e.g., room temperature). In one embodiment, theanti-solvent addition is conducted at an elevated temperature (e.g., 20°C., 25° C., 30° C., 35° C., 40° C., 45° C., 50° C., 55° C., or 60° C.,or higher). In one embodiment, the anti-solvent addition is conducted atnormal pressure (e.g., atmospheric pressure). In one embodiment, theanti-solvent addition is conducted at a reduced pressure (e.g., 0.9,0.8, 0.7, 06, 0.5, 0.4, 0.3, 0.2, or 0.1 of the atmospheric pressure, orlower).

In one embodiment, the crystalline form of the present invention isprepared by vapor sweeping. In one embodiment,2-(6-amino-9H-purin-9-yl)-5-(((3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diolis added to a container (e.g., a vial). In one embodiment, the containeris placed inside a larger container. In one embodiment, a solvent isadded to the larger container and the larger container is sealed.

In one embodiment, the solvent is volatile and forms a vapor in thelarger container. In one embodiment, the solvent is a nitrile, analcohol, THF, methyl THF, an ester or a combination thereof. In oneembodiment, the solvent is acetonitrile, THF, methyl THF, acetonitrile,ethyl acetate, methanol, or a combination thereof.

In one embodiment, the vapor sweeping is conducted over a period of 6hours, 12 hours, 18 hours, 24 hours, 48 hours, 72 hours, 96 hours, or120 hours or 6 days, 7 days, 10 days, 14 days, or more. In oneembodiment, the vapor sweeping is conducted over a period sufficient toallow the solvent vapor to interact with2-(6-amino-9H-purin-9-yl)-5-(((3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol.

In one embodiment, the vapor sweeping is conducted under ambienttemperature (e.g., room temperature). In one embodiment, the vaporsweeping is conducted at an elevated temperature (e.g., 20° C., 25° C.,30° C., 35° C., 40° C., 45° C., 50° C., 55° C., or 60° C., or higher).In one embodiment, the vapor sweeping is conducted at normal pressure(e.g., atmospheric pressure). In one embodiment, the vapor sweeping isconducted at a reduced pressure (e.g., 0.9, 0.8, 0.7, 06, 0.5, 0.4, 0.3,0.2, or 0.1 of the atmospheric pressure, or lower).

In one embodiment, the crystalline form of the present invention isprepared by vapor diffusion. In one embodiment,2-(6-amino-9H-purin-9-yl)-5-(((3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diolis dissolved in a solvent in a container (e.g., a vial). In oneembodiment, the container is placed inside a larger container. In oneembodiment, an anti-solvent is added to the larger container and thelarger container is sealed. In one embodiment, the anti-solvent isvolatile.

In one embodiment, the vapor diffusion is conducted over a period of 6hours, 12 hours, 18 hours, 24 hours, 48 hours, 72 hours, 96 hours, or120 hours or 6 days, 7 days, 10 days, 14 days, or more. In oneembodiment, the vapor diffusion is conducted over a period sufficient toallow the anti-solvent vapor to interact with2-(6-amino-9H-purin-9-yl)-5-(((3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol.

In one embodiment, the vapor diffusion is conducted under ambienttemperature (e.g., room temperature). In one embodiment, the vapordiffusion is conducted at an elevated temperature (e.g., 20° C., 25° C.,30° C., 35° C., 40° C., 45° C., 50° C., 55° C., or 60° C., or higher).In one embodiment, the vapor diffusion is conducted at normal pressure(e.g., atmospheric pressure). In one embodiment, the vapor diffusion isconducted at a reduced pressure (e.g., 0.9, 0.8, 0.7, 06, 0.5, 0.4, 0.3,0.2, or 0.1 of the atmospheric pressure, or lower).

In yet another aspect, the present invention relates to a compound offormula I:

or a salt or solvate thereof, wherein:

R_(a), R_(b), R_(c), and R_(d) are each independently -M₂-T₂;

M₂ is a bond, S(O)₂, S(O), S, C(O), C(O)O, O, O—C₁-C₄ alkyl linker,C₁-C₄ alkyl linker, NH, or NR_(t);

R_(t) is C₁-C₆ alkyl;

T₂ is H, halogen, or R_(S4);

R_(S4) is C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₃-C₆ cycloalkyl,C₆-C₁₀ aryl, 4 to 8-membered heterocycloalkyl, or 5 to 10-memberedheteroaryl;

R_(e) and Rr are each independently H or C₁-C₆ alkyl; and

x is 1, 2, 3, 4, 5, or 6,

wherein each of O—C₁-C₄ alkyl linker, C₁-C₄ alkyl linker, R_(t), andR_(S4) is optionally substituted with one or more substituents selectedfrom the group consisting of halogen, hydroxyl, carboxyl, cyano, C₁-C₆alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ alkoxyl, amino, mono-C₁-C₆alkylamino, di-C₁-C₆ alkylamino, C₃-C₈ cycloalkyl, C₆-C₁₀ aryl, 4 to6-membered heterocycloalkyl, and 5 to 6-membered heteroaryl.

In one embodiment, R_(a). R_(b), R_(c), and R_(d) are each -M₂-T₂,wherein M₂ is a bond; and T₂ is H.

In one embodiment, one of R_(a), R_(b), R_(e), and R_(d) is -M₂-T₂,wherein M₂ is a bond and T₂ is H; and the rest of R_(a), R_(b), R_(c),and R_(d) are each independently -M₂-T₂; wherein M₂ is a bond, S(O)₂,S(O), S, C(O), C(O)O, O, O—C₁-C₄ alkyl linker, C₁-C₄ alkyl linker, NH,or NR; and T₂ is H, halogen, or R_(S4).

In one embodiment, two of R_(a). R_(b), R_(e), and R_(d) is -M₂-T₂,wherein M₂ is a bond and T₂ is H; and the rest of R_(a), R_(b), R_(c),and R_(d) are each independently -M₂-T₂; wherein M₂ is a bond, S(O)₂,S(O), S, C(O), C(O)O, O, O—C₁-C₄ alkyl linker, C₁-C₄ alkyl linker, NH,or NR; and T₂ is H, halogen, or R_(S4).

In one embodiment, three of R_(a), R_(b), R_(c), and R_(d) is -M₂-T₂,wherein M₂ is a bond and T₂ is H; and the rest of R_(a), R_(b), R_(e),and R_(d) is -M₂-T₂; wherein M₂ is a bond, S(O)₂, S(O), S, C(O), C(O)O,O, O—C₁-C₄ alkyl linker, C₁-C₄ alkyl linker, NH, or NR_(t); and T₂ is H,halogen, or R_(S4).

In one embodiment, R_(a), R_(c), and R_(d) are each -M₂-T₂, wherein M₂is a bond and T₂ is H; and R_(b) is -M₂-T₂; wherein M₂ is a bond; T₂ isR_(S4); and R_(S4) is C₁-C₆ alkyl. In one embodiment, R_(S4) is t-butyl.

In one embodiment, x is 2.

In one embodiment, R_(e) and Rr are each H.

In one embodiment, the present invention relates to a compound havingthe following structure:

or a salt or solvate thereof.

The present invention relates to pharmaceutically acceptable salts of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol or a hydratethereof. In one embodiment, the present invention relates to mono-saltsfrom(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diolor a hydrate thereof and 1,5-naphthalene disulfonic acid, H₂SO₄, H₃PO₄,L-tartaric acid and citric acid which may be obtained from direct saltformation. In one embodiment, the present invention relates tomono-salts from(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diolor a hydrate thereof and p-toluene sulfonic acid, methane sulfonic acid,HCl, maleic acid and L-malic acid salt which may be obtained fromfreeze-drying. In one embodiment, the salts are obtained as amorphoussolids. In one embodiment, the present invention relates to mono-saltsfrom(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diolor a hydrate thereof and malonic acid, fumaric acid, galactaric acid andlactobionic acid. In one embodiment, the salts are formed in gums.

In one embodiment, the present invention relates to hemi-salts from(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diolor a hydrate thereof and maleic acid. In one embodiment, the salt isisolated as a gum. In one embodiment, the present invention relates tobis-salts from(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diolor a hydrate thereof and p-toluene sulfonic acid, methane sulfonic acid,and maleic acid. In one embodiment, the salt is isolated as a gum.

The present invention relates to a process for preparing(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol:

or a salt or hydrate thereof, comprising at least one step, or at leasttwo steps selected from the group consisting of:

(1) reacting9-((3aR,4R,6R,6aR)-6-(aminomethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-purin-6-aminewith acetone to yield9-((3aR,4R,6R,6aR)-6-((isopropylamino)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-purin-6-amine;

(2) reacting9-((3aR,4R,6R,6aR)-6-((isopropylamino)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-purin-6-aminewith 3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutanoneto yield9-((3aR,4R,6R,6aR)-6-(((3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-purin-6-amine;and

(3) converting9-((3aR,4R,6R,6aR)-6-(((3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-purin-6-amineto(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-(((3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol.

The present invention relates to a process for preparing(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol:

or a salt or hydrate thereof, comprising the steps of:

(1) reacting9-((3aR,4R,6R,6aR)-6-(aminomethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-purin-6-aminewith acetone to yield9-((3aR,4R,6R,6aR)-6-((isopropylamino)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-purin-6-amine;

(2) reacting9-((3aR,4R,6R,6aR)-6-((isopropylamino)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-purin-6-aminewith 3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutanoneto yield9-((3aR,4R,6R,6aR)-6-(((3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-purin-6-amine;and

(3) converting9-((3aR,4R,6R,6aR)-6-(((3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-purin-6-amineto(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-(((3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol.

In one embodiment, step (1) comprises a solvent. In one embodiment, thesolvent comprises an alcohol. In one embodiment, the solvent comprisesmethanol. In one embodiment,9-((3aR,4R,6R,6aR)-6-(aminomethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-purin-6-amineis dissolved in the solvent. In one embodiment, acetone is mixed in thesolvent. In one embodiment, stirring is performed to facilitate thedissolvation of9-((3aR,4R,6R,6aR)-6-(aminomethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-purin-6-amine.In one embodiment, the dissolvation is conducted under ambienttemperature (e.g., room temperature).

In one embodiment, step (1) further comprises a carboxylic acid. In oneembodiment, the carboxylic acid is acetic acid.

In one embodiment, step (1) further comprises a reducing agent. In oneembodiment, the reducing agent is a borohydride. In one embodiment, theborohydride is sodium triacetoxy borohydride (STAB). In one embodiment,the reducing agent is added to the reaction after9-((3aR,4R,6R,6aR)-6-(aminomethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-purin-6-amineand/or acetone is dissolved in the solvent. In one embodiment, thereducing agent is added to the reaction in multiple portions. In oneembodiment, the solution comprising9-((3aR,4R,6R,6aR)-6-(aminomethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-purin-6-amineand/or acetone is cooled before the reducing agent is added. In oneembodiment, the solution is cooled to a temperature below the ambienttemperature (e.g., room temperature). In one embodiment, the solution iscooled to a temperature below 30° C., 25° C., or 20° C. In oneembodiment, the solution is cooled to 16-18° C.

In one embodiment, the reaction in step (1) is conducted under ambienttemperature (e.g., room temperature). In one embodiment, the reaction isconducted at a temperature below 50° C., 40° C., 35° C., 30° C., 25° C.,or 20° C. In one embodiment, the reaction is conducted at 20-25° C.

In one embodiment, step (1) may further comprise concentrating theresulting9-((3aR,4R,6R,6aR)-6-((isopropylamino)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-purin-6-aminebefore step (2). In one embodiment, the concentration is achievedthrough vacuum evaporation of acetone and the solvent. In oneembodiment,9-((3aR,4R,6R,6aR)-6-((isopropylamino)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-purin-6-amineis filtered before step (2).

In one embodiment, the yield of9-((3aR,4R,6R,6aR)-6-((isopropylamino)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-purin-6-aminethrough step (1) is at least 50%, 60%, 70%, 80%, 90%, 95%, or 99%. Inone embodiment, the yield is at least 90%. In one embodiment, the yieldis at least 95%.

In one embodiment, acetonitrile is added to9-((3aR,4R,6R,6aR)-6-((isopropylamino)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-purin-6-aminefrom step (1), and the mixture is used in step (2) for further reaction.

In one embodiment, step (2) further comprises a carboxylic acid. In oneembodiment, the carboxylic acid is acetic acid.

In one embodiment, step (2) is conducted under an inert gas. In oneembodiment, the inert gas is nitrogen.

In one embodiment, step (2) further comprises a reducing agent. In oneembodiment, the reducing agent is a borohydride. In one embodiment, theborohydride is STAB. In one embodiment, the reducing agent is added tothe mixture of3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutanone and9-((3aR,4R,6R,6aR)-6-((isopropylamino)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-purin-6-amine.In one embodiment, the reducing agent is added to the reaction inmultiple portions. In one embodiment, the mixture of3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutanone and9-((3aR,4R,6R,6aR)-6-((isopropylamino)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-purin-6-amineforms a slurry. In one embodiment, the slurry is heated before STAB isadded to the slurry. In one embodiment, the slurry is heated to 40° C.,45° C., 50° C., 55° C., or 60° C., or higher. In one embodiment, theslurry is heated to 55° C.

In one embodiment, the reaction is stirred for a period of at least 2hours, 4 hours, 8 hours, 12 hours, 18 hours, 24 hours, or 48 hours. Inone embodiment, the reaction is stirred for 14-16 hours.

In one embodiment, the reaction is conducted at a temperature up to 40°C., 45° C., 50° C., 55° C., or 60° C., or higher. In one embodiment, thereaction is conducted at a temperature of 55° C. In one embodiment, thereaction mixture is cooled. In one embodiment, the reaction mixture iscooled to room temperature.

In one embodiment, water is added to the reaction mixture after themixture is cooled.

In one embodiment, step (2) may further comprise concentrating theresulting9-((3aR,4R,6R,6aR)-6-(((3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-purin-6-aminebefore step (3). In one embodiment, the concentration removesacetonitrile.

In one embodiment, the pH of the reaction mixture comprising9-((3aR,4R,6R,6aR)-6-(((3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-purin-6-amineis adjusted with a base (e.g., a hydroxide, such as sodium hydroxide).In one embodiment, the pH is adjusted to about 10. In one embodiment,the aqueous layer is removed after the pH adjustment and the organiclayer is concentrated before step (3).

In one embodiment, the yield of9-((3aR,4R,6R,6aR)-6-(((3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-purin-6-aminethrough step (2) is at least 50%, 60%, 70%, 80%, 90%, 95%, or 99%. Inone embodiment, the yield is at least 80%.

In one embodiment, step (3) is conducted under an inert gas. In oneembodiment, the inert gas is nitrogen.

In one embodiment, step (3) comprises a solvent. In one embodiment, thesolvent comprises an alcohol. In one embodiment, the solvent comprisesmethanol. In one embodiment, step (3) comprises hydrochloric acid. Inone embodiment,9-((3aR,4R,6R,6aR)-6-(((3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-purin-6-amineis mixed with hydrochloric acid and methanol to form a solution. In oneembodiment, the reaction solution is heated to 40° C., 45° C., 50° C.,55° C., or 60° C., or higher. In one embodiment, the reaction solutionis heated to 45° C. In one embodiment, the reaction solution is kept atambient temperature (e.g., room temperature) after the heating.

In one embodiment,9-((3aR,4R,6R,6aR)-6-(((3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-purin-6-amineis converted to(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-(((3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diolafter the heating. In one embodiment, at least 50%, 60%, 70%, 80%, 90%,95%, or 99% of9-((3aR,4R,6R,6aR)-6-(((3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-purin-6-amineis converted. In one embodiment, at least 90% of9-((3aR,4R,6R,6aR)-6-(((3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-purin-6-amineis converted.

In one embodiment, step (3) further comprises adjusting the pH of thereaction solution with a base (e.g., a hydroxide, such as sodiumhydroxide, and a bicarbonate, such as sodium bicarbonate). In oneembodiment, after the pH adjustment the organic layer is concentrated.

The process of the present invention may further comprise step (4):recrystallizing(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-(((3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diolto yield(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diolor a salt of hydrate thereof.

In one embodiment, step (4) further comprises stirring. In oneembodiment, a slurry is formed in step (4). In one embodiment, a seedcrystal is added to the slurry.

In one embodiment, the slurry is heated to 50° C., 55° C., 60° C., 65°C., 70° C., or 75° C., or higher. In one embodiment, the slurry isheated to 75° C. In one embodiment, the slurry is cooled to 25-30° C.after the heating and stirred for 8 hours, 12 hours, 18 hours, or 24hours.

In one embodiment, the heating, cooling, and stirring of the slurry arerepeated.

In one embodiment, the process of the present invention is advantageousas compared to other processes in that the process of the presentinvention produces(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol at a large commercialscale. In one embodiment, the process of the present invention is ableto process at least 100 g, 200 g, 500 g, 1 kg, 2 kg, 5 kg, 10 kg, 20 kg,50 kg, 100 kg, 200 kg, 500 kg, or 1000 kg, or more(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol, without the need toscale up. In one embodiment, the process of the present invention isadvantageous as compared to other processes in that the process of thepresent invention produces(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol at a high purity suchthat cumbersome purification (e.g., column chromatography, filtration,extraction, phase separation, and solvent evaporation) is not needed. Inone embodiment, the process of the present invention is able to process(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol at a purity of at least75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 99.5%, or higher. In oneembodiment, the process of the present invention is advantageous ascompared to other processes in that the process of the present inventionproduces(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol with little or noneimpurity (e.g., the trans-isomer thereof or toxic impurity). In oneembodiment, the impurity produced in the process of the presentinvention, even if produced, is easy to be separated from(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol without any cumbersomepurification (e.g., column chromatography, filtration, extraction, phaseseparation, and solvent evaporation). In one embodiment, the process ofthe present invention is advantageous as compared to other processes inthat the process of the present invention uses less or none catalyst(e.g., metal catalyst, such as transition metal catalyst) such thatminimum or none separation is needed to remove the catalyst. In oneembodiment, the process of the present invention is advantageous ascompared to other processes in that the process of the present inventionproduces(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol at a significantlyreduced cost, due to its high yield, high purity, little or no impurity,reduced amount of or no catalyst, or a combination thereof.

The present invention relates to a process for preparing3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutanone:

or a salt thereof, comprising at least one step, at least two steps, atleast three steps, at least four steps, or at least five steps, selectedfrom the group consisting of:

(1) converting pent-4-enoic acid to benzyl pent-4-enoate;

(2) converting benzyl pent-4-enoate to benzyl3-(2,2-dichloro-3-oxo-cyclobutyl)propanoate;

(3) converting benzyl 3-(2,2-dichloro-3-oxo-cyclobutyl)propanoate tobenzyl 3-(3-oxo-cyclobutyl)propanoate;

(4) converting benzyl 3-(3-oxo-cyclobutyl)propanoate to3-(3-oxo-cyclobutyl)propanoic acid;

(5) reacting 3-(3-oxocyclobutyl)propanoic acid with4-tert-butyl-2-nitroaniline to yieldN-(4-tert-butyl-2-nitrophenyl)-3-(3-oxo-cyclobutyl)propanamide; and

(6) convertingN-(4-tert-butyl-2-nitrophenyl)-3-(3-oxo-cyclobutyl)propanamide to3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutanone.

The present invention relates to a process for preparing3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutanone:

or a salt thereof, comprising the steps of:

(1) converting pent-4-enoic acid to benzyl pent-4-enoate;

(2) converting benzyl pent-4-enoate to benzyl3-(2,2-dichloro-3-oxo-cyclobutyl)propanoate;

(3) converting benzyl 3-(2,2-dichloro-3-oxo-cyclobutyl)propanoate tobenzyl 3-(3-oxo-cyclobutyl)propanoate;

(4) converting benzyl 3-(3-oxo-cyclobutyl)propanoate to3-(3-oxo-cyclobutyl)propanoic acid;

(5) reacting 3-(3-oxocyclobutyl)propanoic acid with4-tert-butyl-2-nitroaniline to yieldN-(4-tert-butyl-2-nitrophenyl)-3-(3-oxo-cyclobutyl)propanamide; and

(6) convertingN-(4-tert-butyl-2-nitrophenyl)-3-(3-oxo-cyclobutyl)propanamide to3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutanone. Inone embodiment,3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutanone saltis a hydrochloride salt.

In one embodiment, step (1) is conducted under an inert gas. In oneembodiment, the inert gas is nitrogen. In one embodiment, step (1)comprises a carbonate (e.g., potassium carbonate). In one embodiment,step (1) further comprises tetrabutylammonium iodide. In one embodiment,the yield of benzyl pent-4-enoate through step (1) is at least 90%.

In one embodiment, step (2) comprises a mixture of benzyl pent-4-enoateand zinc-copper in diethyl ether and 1,2-dimethoxyethane. In oneembodiment, trichloroacetyl chloride is added to the mixture. In oneembodiment, the mixture is stirred at about 50° C. In one embodiment,the mixture is cooled after the stirring. In one embodiment, step (2)further comprises washing and concentrating the organic layer comprisingbenzyl 3-(2,2-dichloro-3-oxo-cyclobutyl)propanoate.

In one embodiment, step (3) comprises treating benzyl3-(2,2-dichloro-3-oxo-cyclobutyl)propanoate with zinc powder a solvent.In one embodiment, the mixture of benzyl3-(2,2-dichloro-3-oxo-cyclobutyl)propanoate and zinc powder is heated to65° C., 70° C., 75° C., 80° C., or 85° C. In one embodiment, the mixtureof benzyl 3-(2,2-dichloro-3-oxo-cyclobutyl)propanoate and zinc powder isheated to 80° C. In one embodiment, the mixture is cooled after theheating. In one embodiment, step (3) further comprises washing andconcentrating the organic layer comprising benzyl3-(3-oxo-cyclobutyl)propanoate.

In one embodiment, step (4) comprises an inert gas. In one embodiment,the inert gas is nitrogen. In one embodiment, step (4) comprises acatalyst. In one embodiment, the catalyst is a palladium catalyst (e.g.,Pd/C). In one embodiment, step (4) comprises hydrogen gas. In oneembodiment, step (4) further comprises washing and concentrating theorganic layer comprising 3-(3-oxo-cyclobutyl)propanoic acid.

In one embodiment, step (5) comprises a solvent. In one embodiment, thesolvent comprises dioxane (e.g., 1,4-dioxane). In one embodiment,3-(3-oxo-cyclobutyl)propanoic acid and 4-tert-butyl-2-nitroaniline aremixed with pyridine and propylphosphonic anhydride. In one embodiment,the mixture is heated to 80° C., 85° C., 90° C., 95° C., 100° C., 105°C., or 110° C. In one embodiment, the mixture of is heated to 100° C. Inone embodiment, the mixture is cooled after the heating. In oneembodiment, step (5) further comprises washing and concentrating theorganic layer comprisingN-(4-tert-butyl-2-nitrophenyl)-3-(3-oxo-cyclobutyl)propanamide.

In one embodiment, step (6) comprises mixingN-(4-tert-butyl-2-nitrophenyl)-3-(3-oxo-cyclobutyl)propanamide with ironpowder in a solvent. In one embodiment, the mixture is heated to 65° C.,70° C., 75° C., 80° C., or 85° C. In one embodiment, the mixture of isheated to 80° C. In one embodiment, the mixture is cooled after theheating. In one embodiment, step (6) further comprises washing andconcentrating the organic layer comprising3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutanone.

The present invention further relates to a process for preparing3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutanone or asalt thereof, comprising at least one step selected from:

(i) converting dicyclohexylammonium 3-(3-oxocyclobutyl)propanoate to3-(3-oxocyclobutyl)propanoyl chloride;

(ii) reacting 3-(3-oxocyclobutyl)propanoyl chloride with4-tert-butyl-2-nitroaniline to yieldN-(4-tert-butyl-2-nitrophenyl)-3-(3-oxo-cyclobutyl)propanamide; and

(iii) convertingN-(4-tert-butyl-2-nitrophenyl)-3-(3-oxo-cyclobutyl)propanamide to3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutanone.

In one embodiment, step (i) comprises the presence of a solvent andoxalyl chloride. In one embodiment, the solvent comprises 1,4-dioxane.In one embodiment, the solvent further comprises dimethylformamide(DMF). In one embodiment, a mixture of dicyclohexylammonium3-(3-oxocyclobutyl)propanoate, 1,4-dioxane and DMF is cooled to about12° C. (e.g., 9° C., 10° C., 11° C., 12° C., 13° C., 14° C., or 15° C.)and oxalyl chloride is slowly added at 12-17° C. (e.g., over 35 min),followed by aging at 18-20° C. for sufficient time (e.g., 18 h) toproduce a reaction mixture containing 3-(3-oxocyclobutyl)propanoylchloride.

In one embodiment, step (ii) comprises a solvent. In one embodiment, thesolvent comprises dioxane (e.g., 1,4-dioxane). In one embodiment,3-(3-oxo-cyclobutyl)propanoic acid and 4-tert-butyl-2-nitroaniline aremixed in 1,4-dioxane, and the mixture is stirred at a suitabletemperature (e.g., 20° C.-40° C.) for sufficient time to yieldN-(4-tert-butyl-2-nitrophenyl)-3-(3-oxo-cyclobutyl)propanamide, forexample, at 20° C. for 1 h, and slowly warmed to 35-40° C. over 4 h andkept at this temperature for 1 h, cooled to 20° C. over 2 h and thenkept at this temperature for 18 h. In one embodiment, step (ii) furthercomprises purifyingN-(4-tert-butyl-2-nitrophenyl)-3-(3-oxo-cyclobutyl)propanamide beforestep (iii).

In one embodiment, step (iii) comprises mixingN-(4-tert-butyl-2-nitrophenyl)-3-(3-oxo-cyclobutyl)propanamide with ironpowder in a solvent. In one embodiment, the mixture is heated to 60° C.,65° C., 70° C., 75° C., 80° C., or 85° C. In one embodiment, the mixtureof is heated for 3 hrs. In one embodiment, the mixture is cooled afterthe heating. In one embodiment, step (iii) further comprises washing andconcentrating the organic layer comprising3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutanone. Inone embodiment, step (iii) further comprises converting3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutanone to itsHCl salt.

In one embodiment, dicyclohexylammonium 3-(3-oxocyclobutyl)propanoateused in step (i) is prepared by reacting 3-(3-oxo-cyclobutyl)propanoicacid with dicyclohexylamine (DCHA) in a solvent. In one embodiment, thesolvent is isopropyl acetate (IPAc). In one embodiment,3-(3-oxo-cyclobutyl)propanoic acid and DCHA (e.g., 1.2 eq.) is mixed inIPAc and the resulting slurry is stirred at about 20-25° C. for, e.g.,18 h to obtain the DCHA salt. In one embodiment, dicyclohexylammonium3-(3-oxocyclobutyl)propanoate is isolated as a white solid, by, e.g.,filtration and washed with IPAc, dried in vacuo at e.g., 45-55° C. witha nitrogen sweep.

In one embodiment, the process of the present invention is shown inScheme 1. The process is a 4-step synthesis including a purificationstep to produce pure(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diolor a hydrate thereof.

The process of the present invention has never been reported in the art.The process is a 4-step synthesis including one purification step. Step1 is the coupling of compound 1 and acetone in the presence of methanol,acetic acid, and sodium triacetoxyborohydride to produce compound 2.Compound 1 may be prepared from commercially available starting materialas described in Journal of Medicinal Chemistry, 1969, 12 (4), 658-662.Step 2 is the coupling of compound 2 and compound 3 (for the preparationof compound 3, see below) in the presence of acetonitrile, acetic acid,and sodium triacetoxyborohydride to produce compound 4. Step 3 is theconversion of compound 4 into compound 5 in the presence of methanol and6N HCl. Step 4 is the recrystallization of compound 5 using a mixture ofan organic solvent (e.g., acetonitrile or isopropyl alcohol) and waterto afford a crystalline form of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diolor a hydrate thereof.

The process of the present invention relates to a process for preparinga crystalline form of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diolor a hydrate thereof comprising a crystalline form of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diolor a hydrate thereof as a synthetic intermediate.

Step 1

Compound 1 and acetone are coupled in the presence of a solvent, anorganic acid, and a reducing agent to produce compound 2. In oneembodiment, the solvent is methanol. In one embodiment, the organic acidis acetic acid. In one embodiment, the reducing agent is sodiumtriacetoxyborohydride. Compound 1 may be prepared from commerciallyavailable starting material as described in Journal of MedicinalChemistry, 1969, 12 (4), 658-662.

Compound 1, acetone, acetic acid, and methanol are added to a reactionvessel at room temperature, the reaction mixture being stirred for 5-10min until all solids are dissolved. In one embodiment, the reactionmixture comprising compound 1, acetone, acetic acid, and methanol iscooled to 16-18° C., and sodium acetoxyborohydride is added over 1-2min. In one embodiment, sodium acetoxyborohydride is added to thereaction mixture comprising compound 1, acetone, acetic acid, andmethanol in 4 equal portions over 2 h, maintaining the batch temperaturebetween 20-25° C. The batch as a solution is stirred at the sametemperature for an additional 1-2 h. In one embodiment, the reactionmixture is concentrated on a rotary evaporator under vacuum to removeall acetone and methanol, and flushed with acetonitrile (4.4 L×2). Someinorganic solids were precipitated out during the concentration.

In one embodiment, solid is removed by filtration, the wet cake iswashed with acetonitrile. The combined filtrate is concentrated andflushed with acetonitrile to give a concentrated oil. In one embodiment,acetonitrile is added to the concentrated oil, the resulting solution isanalyzed by HPLC assay. In one embodiment, the resulting solution ispassed through an in-line filter (10 micron) to the reaction vessel (50L size) to be used in Step 2. The line is rinsed with acetonitrile tomaintain an appropriate volume of solvent.

Step 2

Compound 2 and compound 3 are coupled in the presence of a solvent, anorganic acid, and a reducing agent to produce compound 4. In oneembodiment, the solvent is acetonitrile. In one embodiment, the organicacid is acetic acid. In one embodiment, the reducing agent is sodiumtriacetoxyborohydride.

Compound 2 and acetonitrile are added to a reaction vessel at roomtemperature. In one embodiment, acetic acid and compound 3 are added tothe reaction vessel at room temperature under nitrogen. In oneembodiment, the reaction mixture comprising compound 2, acetonitrile,acetic acid, and compound 3 is heated to 55° C. In one embodiment,sodium triacteoxyborohydride is added to the reaction mixture over 1-2minutes. In one embodiment, additional compound 3 is added to thereaction mixture in 3 portions over 4 hours and additional sodiumtriacteoxyborohydride in 9 portions over 5 hours. In one embodiment, thereaction mixture is stirred at 55° C. for 14-16 h. In one embodiment,the reaction mixture is cooled to room temperature and water is addedover 1-2 min with stirring. In one embodiment the reaction mixture(batch) is warmed to room temperature and the bottom aqueous layer isremoved. In one embodiment, the organic layer was concentrated to removemost of the acetonitrile. Methyl tert-butyl ether (MTBE) and methanolare added. The resulting solution is cooled to 5-10° C. In oneembodiment, 3N NaOH is added slowly with stirring to adjust aqueouslayer pH from 6 to 10, while the mixture temperature was maintained at25-30° C. When the aqueous layer reached to pH 10, the stirring wasstopped and the layers were separated. In one embodiment, the aqueouslayer is removed, and the organic layer is washed with 5% NaHCO₃. Theaqueous layer was removed again, the used aqueous layer pH should be 9.In one embodiment, the organic layer is concentrated and flushed withmethanol to remove all MTBE. The resulting thick oil is diluted withMeOH to afford a clear light brown solution. The solution was ready forStep 3 without further purification.

Step 3

Compound 4 is converted into compound 5 in the presence of a solvent andan acid. In one embodiment, the solvent is methanol. In one embodiment,the acid is hydrochloric acid (HCl). In one embodiment, the acid is 6Nhydrochloric acid.

Compound 4, methanol, and 6N HCl are added to a reaction vessel. In oneembodiment, the reaction mixture comprising compound 4, methanol, and 6NHCl are heated at 45° C. for 7-9 h. In one embodiment, the reactionmixture is maintained at ambient temperature for 12-16 h. In oneembodiment, the reaction mixture is cooled to 5-10° C., and 3N NaOH isadded slowly keeping the temperature at the range of 25-30° C. Theaqueous layer pH should be at the range of 3-4. In one embodiment, MTBEis added with stirring. 3N NaOH is added slowly with stirring keepingthe temperature at the same range of 25-30° C. The target pH should be10. In one embodiment, saturated aqueous NaHCO₃ is added with stirring.The aqueous layer pH should be around 9. In one embodiment, the layersare separated; the aqueous layer is extracted with MTBE and MeOH once.The combined organic layers are concentrated and the concentratedresidue is flushed with acetonitrile to remove all MTBE and methanol toafford a sticky residue. In one embodiment, the sticky residue is mixedwith 3:1 MeCN/water and warmed to 45° C., to obtain a clear solution. Inone embodiment, the solution is transferred to a reaction vessel via aninline filter (Polycap 36 TC, 1.0 micron) to remove all fibers and dust.In one embodiment, the line is rinsed with 3:1 MeCN/water. The solutionin the vessel was ready for Step 4 without further purification.

Step 4

Compound 5 is recrystallized using a solvent to afford a crystallineform of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diolhydrate. In one embodiment, the solvent is a mixture of water and anorganic solvent (e.g., acetonitrile or isopropyl alcohol). In oneembodiment, the solvent is a mixture of acetonitrile and water. In oneembodiment, the solvent is a 3:1 mixture of acetonitrile and water. Inone embodiment, the solvent is a mixture of isopropyl alcohol and water.In one embodiment, the solvent is a 9:1 mixture of isopropyl alcohol andwater.

Compound 5 and 3:1 MeCN/water are added to a reaction vessel. In oneembodiment, the reaction mixture (batch) comprising compound 5 and 3:1MeCN/water is maintained at 30° C. In one embodiment, the reactionmixture is heated to 45° C. to dissolve any precipitate and cooled backto 30° C. In one embodiment, solid seeds of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diolhydrate are added at 30° C. with stirring. In one embodiment, a white,thin slurry is generated within 30 min, and the mixture is stirred at25-30° C. for 1 h. In one embodiment, the slurry is heated to 75° C. andstirred at the same temperature for 1-2 h. In one embodiment, the slurryis cooled slowly back to 30° C. over 4-5 h and stirred at the sametemperature for an additional 12-16 h. In one embodiment, the slurry iscooled to room temperature. After being stirred at the same temperaturefor 2-3 h, the slurry is filtered through coarse porosity sintered glassfunnel to afford a wet cake. In one embodiment, the wet cake is washedwith 3:1 MeCN/water to afford a solid. In one embodiment, the solid isdried in air at room temperature with a vacuum suction for 2-3 h toremove most of solvent to afford a partially dried solid. In oneembodiment, the solid has a cis/trans isomer ratio of 97:3.

In one embodiment, the partially dried solid and 3:1 MeCN/water is addedto a reaction vessel to afford a mixture as a slurry. In one embodiment,the slurry is heated to 75° C. and stirred at the same temperature for1-2 h. In one embodiment, the mixture is cooled slowly to 30° C. over 6h, and stirred at the same temperature for an additional 12-16 h. In oneembodiment, the mixture is cooled to room temperature. After beingstirred at the same temperature for 2-3 h, the slurry is filteredthrough coarse porosity sintered glass funnel to afford a wet cake. Inone embodiment, the wet cake is washed with 3:1 MeCN/water to afford asolid. In one embodiment, the solid is dried in air at room temperaturewith a vacuum suction for 20-30 h to remove solvent. In one embodiment,the solids are occasionally turned over to speed up the drying process.When the weight of batch remained as constant it was considered to bedry. In one embodiment, the resulting solid has a cis/trans isomer ratioof >99:1 (e.g., 99.2:0.8).

In one embodiment, the solid with a cis/trans isomer ratio of >99:1undergoes a further recrystallization by e.g., mixing the solid with anisopropyl alcohol (IPA)/water mixture. In one embodiment, the solid ismixed with 9:1 IPA/water and heated to 65° C. until dissolution. In oneembodiment, the solution is filtered through a fine porosity sinteredglass funnel and the filtrate is heated to 45° C. to form a slurry,which is stirred at 45° C. for 2 h while DI water is slowly added, e.g.,over 12 h, e.g., by a syringe pump. In one embodiment, the resultingmixture is kept at 45° C. for, e.g., 5 h, and cooled linearly to 15° C.over 2 h. In one embodiment, the recrystallization product is isolatedby filtration and washed with 1:1 IPA-water followed by drying in vacuoat 40° C. to constant weight.

In one embodiment, the above partially dried solid (e.g., with acis/trans isomer ratio of 97:3) is mixed with 9:1 IPA/water (instead ofmixing with 3:1 MeCN/water) and heated to 65° C. until dissolution. Inone embodiment, the solution is filtered through a fine porositysintered glass funnel and the filtrate is heated to 45° C. to form aslurry, which is stirred at 45° C. for 2 h while DI water is addedslowly, e.g., over 12 h, e.g., by a syringe pump. In one embodiment, theresulting mixture is kept at 45° C. for, e.g., 5 h, and cooled linearlyto 15° C. over 2 h. In one embodiment, the recrystallization product isisolated by filtration and washed with 1:1 IPA-water followed by dryingin vacuo at 40° C. to constant weight. In one embodiment, therecrystallization product has a cis/trans isomer ratio of about 98 to 1.

The process of the present invention relates to a process for preparinga crystalline form of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diolor a hydrate thereof comprising a crystalline form of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diolor a hydrate thereof as a synthetic intermediate.

In one embodiment, the present invention relates to a process forpreparing a crystalline form of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diolor a hydrate thereof, comprising the step of

recrystallizing(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-(((3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diolin a first solvent to yield(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diolor a hydrate thereof.

In one embodiment, the present invention relates to a process forpreparing a crystalline form of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diolor a hydrate thereof, comprising the steps of

recrystallizing(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-(((3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diolin a first solvent to yield(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol or a hydrate thereof,and

converting9-((3aR,4R,6R,6aR)-6-(((3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-purin-6-aminein the presence of a second solvent and an acid to(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-(((3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol.

In one embodiment, the present invention relates to a process forpreparing a crystalline form of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diolor a hydrate thereof, comprising the steps of recrystallizing(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-(((3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diolin a first solvent to yield(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diolor a hydrate thereof, converting9-((3aR,4R,6R,6aR)-6-(((3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-purin-6-aminein the presence of a second solvent and an acid to(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-(((3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol,and coupling9-((3aR,4R,6R,6aR)-6-((isopropylamino)methyl)-2,2-dimethyltetrahydrofluro[3,4-d][1,3]dioxol-4-yl)-9H-purin-6-aminewith 3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutanoneor a salt thereof in the presence of a third solvent, a first organicacid, and a first reducing agent to yield9-((3aR,4R,6R,6aR)-6-(((3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)-2,2-dimethyltetrahydrofluro[3,4-d][1,3]dioxol-4-yl)-9H-purin-6-amine.

In one embodiment, the present invention relates to a process forpreparing a crystalline form of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diolor a hydrate thereof, comprising the steps of

recrystallizing(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-(((3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diolin a first solvent to yield(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diolor a hydrate thereof,

converting9-((3aR,4R,6R,6aR)-6-(((3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-purin-6-aminein the presence of a second solvent and an acid to(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-(((3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol,and

coupling9-((3aR,4R,6R,6aR)-6-((isopropylamino)methyl)-2,2-dimethyltetrahydrofluro[3,4-d][1,3]dioxol-4-yl)-9H-purin-6-aminewith 3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutanoneor a salt thereof in the presence of a third solvent, a first organicacid, and a first reducing agent to yield9-((3aR,4R,6R,6aR)-6-(((3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)-2,2-dimethyltetrahydrofluro[3,4-d][1,3]dioxol-4-yl)-9H-purin-6-amine.

coupling9-((3aR,4R,6R,6aR)-6-(aminomethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-purin-6-aminewith acetone in the presence of a fourth solvent, a second organic acid,and a second reducing agent to yield9-((3aR,4R,6R,6aR)-6-((isopropylamino)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-purin-6-amine.

In one embodiment, the present invention relates to a process forpreparing a crystalline form of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diolor a hydrate thereof, where the crystalline form is the free base of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-dioltrihydrate.

In one embodiment, the present invention relates to a process forpreparing a crystalline form of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diolor a hydrate thereof, further comprising one or more additional steps of

recrystallizing(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-(((3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diolin a first solvent to yield(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol or a hydrate thereof.

In one embodiment, the present invention relates to a process forpreparing a crystalline form of Form A of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diolhydrate.

In one embodiment, the present invention relates to a process forpreparing a crystalline form of Form B of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-dioltrihydrate.

In one embodiment, the present invention relates to a process forpreparing a crystalline form of Form C of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diolanhydrate.

In one embodiment, the present invention relates to a process forpreparing a crystalline form of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diolor a hydrate thereof having a purity greater than 98.0%

In one embodiment, the present invention relates to a process forpreparing a crystalline form of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diolor a hydrate thereof having a purity greater than 98.5%

In one embodiment, the present invention relates to a process forpreparing a crystalline form of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diolor a hydrate thereof having a purity greater than 99.0%

In one embodiment, the present invention relates to a process forpreparing a crystalline form of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diolor a hydrate thereof containing a total of less than 2.0% of one or moreimpurities.

In one embodiment, the present invention relates to a process forpreparing a crystalline form of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diolor a hydrate thereof containing a total of less than 1.5% of one or moreimpurities.

In one embodiment, the present invention relates to a process forpreparing a crystalline form of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diolor a hydrate thereof containing a total of less than 1% of one or moreimpurities.

In one embodiment, the present invention relates to a process forpreparing a crystalline form of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diolor a hydrate thereof containing(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1s,3R)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diolor a hydrate thereof as an impurity.

In another aspect, the invention features a composition comprising(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol or a hydrate thereofand the trans-isomer thereof or a hydrate of the trans-isomer, whereinthe content of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol or a hydrate thereof inthe composition is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%,or 99.5%, or higher. In one embodiment, the composition contains lessthan 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, or 0.5% or lower of thetrans-isomer or a hydrate of the trans-isomer.

The present invention is also directed to a process for preparing3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutanone or asalt thereof, having the chemical structure:

The present invention is directed to a process for preparing3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutanone or asalt thereof. The process of the present invention is a six-stepsynthesis and is shown in Scheme 2.

The process of the present invention has never been reported in the art.The process is a 6-step synthesis. Step 1 is the conversion of compound6 into compound 7 in the presence of acetone, potassium carbonate,tetrabutylammonium iodide, and benzyl bromide. Step 2 is the conversionof compound 7 into compound 8 in the presence of diethyl ether,1,2-dimethoxyethane, zinc-copper couple, and trichloroacetyl chloride.Step 3 is the conversion of compound 8 into compound 9 in the presenceof acetic acid and zinc powder. Step 4 is the conversion of compound 9into compound 10 in the presence of ethyl acetate, Pd/C, and hydrogengas. Step 5 is the coupling of compound 10 and compound 11 in thepresence of 1,4-dioxane, pyridine, and propylphosphonic anhydride. Step6 is the conversion of compound 12 into compound 3 in the presence ofacetic acid and iron powder.

The present invention is directed to a process for preparing3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutanone or asalt thereof, as a synthetic intermediate.

Step 1

Compound 6 is converted into compound 7 in the presence of a solvent,metal carbonate, tetraalkylammonium salt, and benzyl bromide. In oneembodiment, the solvent is acetone. In one embodiment, the metalcarbonate is potassium carbonate. In one embodiment, thetetraalkylammonium salt is tetrabutylammonium iodide.

Compound 6, benzyl bromide, and acetone are added to a reaction vessel.In one embodiment, potassium carbonate tetrabutylammonium iodide areadded to the reaction mixture comprising compound 6, benzyl bromide, andacetone to afford a suspension. In one embodiment, the suspension isstirred at room temperature for two days to afford a solid. In oneembodiment, the solid is filtered and washed with acetone. In oneembodiment, the organic solvent is evaporated and the resulting residueis dissolved in ethyl acetate, washed with 2M HCl, saturated NaHCO₃,brine, dried over Na₂SO₄, filtered and concentrated to yield a colorlessoil. In one embodiment, no purification is required.

Step 2

Compound 7 is converted into compound 8 in the presence of a solvent,zinc-copper couple, and trichloroacetyl chloride. In one embodiment, thesolvent is an ethereal solvent. In one embodiment, the solvent is amixture of diethyl ether and 1,2-dimethoxyethane.

In Step 2, zinc-copper couple in diethyl ether and 1,2-dimethoxyethaneis treated dropwise with trichloroacetyl chloride to form a mixture. Inone embodiment, the mixture is stirred at 50° C. for 3 days. In oneembodiment, the mixture is cooled to room temperature, celite is added,and the mixture is stirred for ˜5 minutes and then filtered through aplug of celite. The resulting solid and celite is washed with TBME. Thecombined organic washings are washed with water, saturated NaHCO₃solution, brine, dried over Na₂SO₄, filtered and concentrated to yield abrown oil. In one embodiment, the brown oil is stirred with 50 ml ofheptane for 10-15 min. Next, the stirring is stopped and the heptanelayer is removed. This is repeated until the brown oil turns into asolid. In one embodiment, the combined heptane layers are concentratedto a yellow oil.

Step 3

Compound 8 is converted into compound 9 in the presence of a solvent andzinc powder. In one embodiment, the solvent is acetic acid.

Compound 8 in acetic acid is treated with small portions of zinc powderat room temperature to form a reaction mixture. In one embodiment, thereaction mixture is stirred at 80° C. for 2 hours. In one embodiment,the reaction mixture is cooled to room temperature, diluted with TBME,filtered, and concentrated in vacuo. For example, heptane is added toremove most of the acetic acid azeotropically to give a viscous liquid.In one embodiment, water is added to the viscous liquid and the mixtureis extracted with ethyl acetate. In one embodiment, the combined organicphase is washed with saturated NaHCO₃, brine, dried over Na₂SO₄,filtered, and concentrated to yield a clear yellow oil.

Step 4

Compound 9 is converted into compound 10 in the presence of a solvent,Pd/C, and hydrogen gas. In one embodiment, the solvent is ethyl acetate.

A solution of compound 9 in ethyl acetate is purged 3 times with N₂before 10% Pd/C is added. The reaction mixture is purged again 3 timeswith N₂ then twice with H₂ before leaving the reaction under anatmosphere of H₂. In one embodiment, the reaction is monitored by LCMSuntil no more sign of starting material is observed. In one embodiment,the reaction is purged 3 times with N₂, filtered through celite, andPd/C is washed 3 times with ethyl acetate. In one embodiment, thecombined organic washes are concentrated to yield a light yellow oil. Inone embodiment, NMR analysis shows clean product and no furtherpurification is required.

Step 5

Compound 10 is coupled to compound 11 in the presence of a solvent, abase, and a coupling reagent to form compound 12. In one embodiment, thesolvent is 1,4-dioxane. In one embodiment, the base is an amine base. Inone embodiment, the amine base is pyridine. In one embodiment, thecoupling reagent is propylphosphonic anhydride. In one embodiment, thepropylphosphonic anhydride is a 50% solution in ethyl acetate.

Compound 10 and compound 11 is dissolved in 1,4-dioxane and pyridine andpropylphosphonic anhydride is added in the form of a 50% solution inethyl acetate at room temperature to form a reaction mixture. In oneembodiment, the reaction mixture is heated to 100° C. and left for 7hrs. In one embodiment, the reaction mixture is cooled to roomtemperature, diluted with ethyl acetate, and washed with 2M NaOH, 2MHCl, and brine and dried over MgSO₄ and concentrated in vacuo to givethe crude product. In one embodiment, the crude product is purified bysilica flash column chromatography using between 100% heptanes to 40%ethyl acetate:60% heptanes as eluent to give the product as a yellowoil.

Step 6

Compound 12 is converted to compound 3 in the presence of a solvent andiron powder. In one embodiment, the solvent is acetic acid.

Compound 12 is dissolved in acetic acid and iron powder is added at roomtemperature to form a reaction mixture. In one embodiment, the reactionmixture is heated to 80° C. and left for 1 hr. In one embodiment, thereaction mixture is cooled to room temperature and filtered through GF(glass fibre) filter paper under suction to give a solid. In oneembodiment, the solid is washed with ethyl acetate. In one embodiment,the solvents are removed in vacuo and the resultant residue is dissolvedin dichloromethane. Saturated Na₂CO₃ solution is added until the mixtureis no longer acidic. In one embodiment, the mixture is filtered throughCelite under suction and the plug washed with dichloromethane. In oneembodiment, the layers are separated and the aqueous layer is extractedwith dichloromethane. The combined organic layers are dried over MgSO₄,filtered and concentrated in vacuo to give the crude product as aresidue. In one embodiment, the product is salted by dissolving theresidue in dichloromethane and adding 2M HCl in ether. For example,after about 30 seconds of swirling the solvent a white precipitate isformed. In one embodiment, the precipitate is filtered under suction,washed with ether and dried under vacuum at 50° C. for 2 hours to givethe pure product, which was pure enough for use without subsequentpurification, as a white powder.

The present invention is also directed to an alternative process forpreparing3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutanone or asalt thereof. The alternative process of the present invention is shownin Scheme 2A (in which Bn=benzyl).

The alternative process presented in Scheme 2A is also a 6-stepsynthesis. Step 1 is the conversion of compound 6 to compound 7 in thepresence of a mixture of water and toluene, potassium iodide,tripotassium phosphate, tetrabutylammonium bromide, and benzyl chloride.Step 2 is the conversion of compound 7 to compound 8 in the presence of1,4-dioxane, zinc-copper couple, and trichloroacetyl chloride. Step 3 isthe conversion of compound 8 to compound 9 in the presence of aceticacid and zinc powder. Step 4 is the conversion of compound 9 to compound10 in the presence of isopropyl acetate, toluene, Pd/C, and hydrogengas, which is followed by conversion of compound 10 to compound 10B inthe presence of dicyclohexylamine (DCHA). Step 5 is the conversion ofcompound 10B to compound 10C in the presence of 1,4-dioxane, DMF, andoxalyl chloride, which is followed by the conversion of compound 10C tocompound 12 in the presence of 4-t-butyl-2-nitroaniline, and1,4-dioxane. Step 6 is the conversion of compound 12 into compound 3 inthe presence of acetic acid and iron powder.

In another aspect, the present invention is directed to a process forpreparing3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutanone or asalt thereof. The process includes one or more of the following 6 steps.

Step 1

Compound 6 is converted into compound 7 in the presence of a solvent, ametal phosphate or metal carbonate, a metal halide, a tetraalkylammoniumsalt, and a benzyl halide (e.g., benzyl chloride or bromide). In oneembodiment, the solvent is a mixture of water and toluene (e.g., with avolume ratio of 1 to 1). In one embodiment, the metal carbonate ispotassium carbonate. In one embodiment, the metal phosphate istripotassium phosphate. In one embodiment, the tetraalkylammonium saltis tetrabutylammonium bromide. In one embodiment, the metal halide ispotassium iodide. In one embodiment, the benzyl halide is benzylchloride.

In one embodiment, compound 6, and then benzyl chloride are added to areaction vessel containing tripotassium phosphate, tetrabutylammoniumbromide, potassium iodide, water and toluene to afford a suspension. Inone embodiment, the suspension is stirred at about 60-70° C. (e.g.,62-65° C.) for sufficient time (e.g., 10-30 hours or about 20 hours) toafford an organic layer comprising compound 7. In one embodiment, thesuspension is stirred at about 60-70° C. (e.g., 62-65° C.) forsufficient time (e.g., 10-30 hours) and then treated with triethylamineand stirred at about 60-70° C. (e.g., 62-65° C.) for sufficient time(e.g., 10-30 hours or about 20 hours) to afford an organic layercomprising compound 7. In one embodiment, the organic layer is washedwith water at an elevated temperature (e.g., 60-70° C., or about 65° C.)and then cooled to about 25° C., dried over Na₂SO₄, and filtered, e.g.,through a pad of Solka Floc 40 (e.g., soaked in an organic solvent suchas toluene). In one embodiment, the organic layer is concentrated toafford a light brown liquid comprising compound 7 in toluene.

Step 2

Compound 7 is converted into compound 8 in the presence of a solvent,zinc-copper couple, and trichloroacetyl chloride. In one embodiment, thesolvent is 1,4-dioxane. In one embodiment, compound 7 in dioxane istreated with Zn—Cu couple at an elevated temperature (e.g., 40-50° C. or45° C.) followed by slow addition of trichloroacetyl chloride at atemperature between 50-80° C. to form a mixture. In one embodiment, uponcompletion of the addition of trichloroacetyl chloride, the mixture isstirred at about 60-65° C. for sufficient time (e.g., 1 hour) to affordcompound 8. In one embodiment, the mixture is cooled to roomtemperature, stirred overnight, and then filtered, e.g., through aSolka-Floc. The resulting solid is washed with dioxane. The combinedorganic washings are then concentrated to yield crude compound 8.

Step 3

Compound 8 is converted into compound 9 in the presence of a solvent andzinc powder. In one embodiment, the solvent is acetic acid (e.g.,glacial acetic acid). In one embodiment, zinc powder (e.g., 6-9μ) isadded in portions into a suspension of compound 8 in acetic acid at anelevated temperature (e.g., 40-90° C.) to form a reaction mixture. Inone embodiment, the reaction mixture is stirred at 60° C. for 0.5 hoursand then at room temperature overnight. In one embodiment, the reactionmixture is then filtered, washed with ethyl acetate and concentrated invacuo. In one embodiment, the residue is partitioned between ethylacetate and water, with the aqueous layer being separated, and theorganic layer being washed with a KHPO₄ solution and then water. Theorganic phase is collected, dried over Na₂SO₄ to yield crude compound 9.In one embodiment, crude compound 9 is purified by eluting through asilica gel with 9:1 hexanes/ethyl acetate.

Step 4

Compound 9 is converted into compound 10 in the presence of a solvent,Pd/C, and hydrogen gas. In one embodiment, the solvent is a mixture ofisopropyl acetate and toluene (e.g., with a volume ratio of 3:1 of IPActo toluene). In one embodiment, 10% Pd/C is added into a solution ofcompound 9 in isopropyl acetate and toluene and the resulting mixture ispurged by alternating vacuum and nitrogen cycles (3×), followed byvacuum and hydrogen gas (3×). In one embodiment, the reaction mixture istreated with hydrogen gas (60 psi) at 20-25° C. for sufficient time(e.g., 20 h) to provide compound 10. In one embodiment, the reactionproduct is washed with IPAc and the filtrate containing compound 10 isstored for the dicyclohexylammonium (DCHA) salt (10B) formation withoutfurther purification. In one embodiment, compound 10 is converted intocompound 10B in the presence of dicyclohexylamine (DCHA) and IPAc. Inone embodiment, DCHA (e.g., 1.2 eq.) is added into an IPAc solution ofcompound 10 and the resulting slurry is stirred at, e.g., about 20-25°C., for, e.g., 18 h. In one embodiment, the resulting product isisolated by filtration and washed with IPAc, dried in vacuo at e.g.,45-55° C. with a nitrogen sweep to yield compound 10B as a white solid.

Step 5

Compound 10B is converted into compound 10C in the presence of a solventand oxalyl chloride. In one embodiment, the solvent comprises1,4-dioxane. In one embodiment, the solvent further comprises DMF. Inone embodiment, a mixture of compound 10B, 1,4-dioxane and DMF is cooledto about 12° C. and oxalyl chloride is slowly added at 12-17° C., e.g.,over 35 min, followed by aging at 18-20° C. for sufficient time, e.g.,18 h to produce a reaction mixture containing 10C. In one embodiment, asolution of 4-t-butyl-2-nitroaniline in 1,4-dioxane is slowly added tothe reaction mixture containing 10C, e.g., over 60 min, at e.g., 15-20°C. In one embodiment, the resulting orange-yellow slurry is stirred at asuitable temperature for sufficient time to yield compound 12, forexample, at 20° C. for 1 h, and slowly warmed to 35-40° C. over 4 h andkept at this temperature for 1 h, cooled to 20° C. over 2 h and thenkept at this temperature for 18 h. In one embodiment, the reactionproduct containing compound 12 is filtered to remove DCHA HCl salt andwashed with 1,4-dioxane, and then the filtrate is combined and thenconcentrated in vacuo at 45° C., flushed and then diluted with aceticacid (AcOH). In one embodiment, the AcOH solution is warmed to 35° C.,and DI water is added over 2 h, and then the mixture is seeded with aseed crystal of compound 12 (e.g., withpurity >80%, >85%, >90%, >92%, >95%, >97%, >98%, >99%, or >99.5%) toform an orange slurry. In one embodiment, the slurry is stirred at30-35° C. for 3 h, and then at 18-20° C. for 14 h before the slurry isfiltered. In one embodiment, the wet cake is washed with 2:3 AcOH/H₂Oand dried in vacuo at 50° C. to yield purified compound 12 as a yellowsolid.

Step 6

Compound 12 is converted to compound 3 in the presence of a solvent andiron powder. In one embodiment, the solvent is acetic acid. In oneembodiment, compound 12 is dissolved in acetic acid and iron powder isadded at an elevated temperature (e.g., 45-67° C.) slowly, e.g., over 1hour, to form a reaction mixture. In one embodiment, the reactionmixture is kept at 65-75° C. for 3 hrs before completion. In oneembodiment, the resulting reaction mixture is then cooled to roomtemperature and kept at this temperature overnight to obtain a slurry.In one embodiment, the slurry is filtered, and the wet cake washed withtoluene, and the combined filtrate is concentrated, and flushed withtoluene to remove most of AcOH and to arrive at crude 3 free base, as athick oil (containing AcOH and toluene). In one embodiment, the crude 3free base is diluted with DCM and neutralized by adding Na₂CO₃ until theaqueous layer pH reaches 7, and the organic layer is then dried overNa₂SO₄, washed with DCM to produce purified 3 free base in DCM. In oneembodiment, the DCM solution is cooled to 0-5° C., and 4 N HCl indioxane is added slowly, e.g., over 1 hour at 5-10° C., and then after˜50% of the HCl is added, the mixture is seeded with a seed crystal ofcompound 3 (e.g., withpurity >80%, >85%, >90%, >92%, >95%, >97%, >98%, >99%, or >99.5%), andthe remaining HCl is added at, e.g., 18-20° C. In one embodiment, theresulting slurry is kept at this temperature for e.g., 17 h, and thenfiltered and washed with DCM. In one embodiment, the wet cake is driedin vacuo (at e.g., 40-45° C.) with a nitrogen sweep to produce compound3 as an off-white solid.

The process of the present invention is an improvement over theprocesses disclosed in the prior art. The preparation of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydro-furan-3,4-diolis disclosed in WO 2012/075381 (referred to herein as the “'381application”).

The process to prepare(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diolas described in the '381 application is depicted in Scheme 3.

The process of the '381 application as outlined in Scheme 3 is a linear9-step synthetic process which includes 6 purification steps. Theprocess of the present application is a convergent synthetic processconsisting of the 4 steps as outlined in Scheme 1 and the 6 steps neededto synthesize compound 3 as outlined in Scheme 2.

The process of the '381 application is significantly different than theprocess of the present invention. The synthetic sequence of the '381application is significantly different than the synthetic sequence ofthe present invention. The structures of the intermediates in thesynthetic sequence of the '381 application are significantly differentthan the structures of the intermediates in the synthetic sequence ofthe present invention. The purification step of the final compound,(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol,of the '381 application is significantly different than the purificationstep performed in the present invention. For example, the '381application uses preparative-HPLC to purify the final compound,(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol.The present invention uses recrystallization to purify the finalcompound,(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol.

The process of the '381 application as outlined in Scheme 3 includes thefollowing synthetic steps: In Step 1, compound 1A is coupled to compound2A in a reductive amination reaction to afford compound 3A. In Step 2,compound 3A is coupled to acetone in a reductive amination reaction toafford compound 4A. In Step 3, the methyl ester of compound 4A isreduced to an aldehyde to afford compound 5A. In Step 4, compound 5A isconverted into compound 6A in a Wittig-type reaction. In Step 5, thealkene of compound 6A is reduced to afford compound 7A. In Step 6, theethyl ester of compound 7A is hydrolyzed to afford compound 8A. In Step7, the carboxylic acid of compound 8A is coupled to an aromatic amine toafford compound 9A. In Step 8, a cyclization reaction converts compound9A into compound 10A. In Step 9, deprotection of compound 10A affords(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol.

As used herein, “compound of the invention” or “compounds of theinvention” may refer to any compounds, or crystalline forms of thepresent invention.

As used herein, “alkyl”, “C₁, C₂, C₃, C₄, C₅ or C₆ alkyl” or “C₁-C₆alkyl” is intended to include C₁, C₂, C₃, C₄, C₅ or C₆ straight chain(linear) saturated aliphatic hydrocarbon groups and C₃, C₄, C₅ or C₆branched saturated aliphatic hydrocarbon groups. For example, C₁-C₆alkyl is intended to include C₁, C₂, C₃, C₄, C₅ and C₆ alkyl groups.Examples of alkyl include, moieties having from one to six carbon atoms,such as, but not limited to, methyl, ethyl, n-propyl, i-propyl, n-butyl,s-butyl, t-butyl, n-pentyl, s-pentyl or n-hexyl.

In certain embodiments, a straight chain or branched alkyl has six orfewer carbon atoms (e.g., C₁-C₆ for straight chain, C₃-C₆ for branchedchain), and in another embodiment, a straight chain or branched alkylhas four or fewer carbon atoms.

As used herein, the term “cycloalkyl” refers to a saturated orunsaturated nonaromatic hydrocarbon mono- or multi-ring system having 3to 30 carbon atoms (e.g., C₃-C₁₀). Examples of cycloalkyl include, butare not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl, cyclooctyl, cyclopentenyl, cyclohexenyl, cycloheptenyl, andadamantyl. The term “heterocycloalkyl” refers to a saturated orunsaturated nonaromatic 5-8 membered monocyclic, 8-12 membered bicyclic,or 11-14 membered tricyclic ring system having one or more heteroatoms(such as O, N, S, or Se). Examples of heterocycloalkyl groups include,but are not limited to, piperazinyl, pyrrolidinyl, dioxanyl,morpholinyl, and tetrahydrofuranyl.

The term “optionally substituted alkyl” refers to unsubstituted alkyl oralkyl having designated substituents replacing one or more hydrogenatoms on one or more carbons of the hydrocarbon backbone. Suchsubstituents can include, for example, alkyl, alkenyl, alkynyl, halogen,hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, amino(including alkylamino, dialkylamino, arylamino, diarylamino andalkylarylamino), acylamino (including alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl,alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.

An “arylalkyl” or an “aralkyl” moiety is an alkyl substituted with anaryl (e.g., phenylmethyl (benzyl)). An “alkylaryl” moiety is an arylsubstituted with an alkyl (e.g., methylphenyl).

As used herein, “alkyl linker” is intended to include C₁, C₂, C₃, C₄, C₅or C₆ straight chain (linear) saturated divalent aliphatic hydrocarbongroups and C₃, C₄, C₅ or C₆ branched saturated aliphatic hydrocarbongroups. For example, C₁-C₆ alkyl linker is intended to include C₁, C₂,C₃, C₄, C₅ and C₆ alkyl linker groups. Examples of alkyl linker include,moieties having from one to six carbon atoms, such as, but not limitedto, methyl (—CH₂—), ethyl (—CH₂CH₂—), n-propyl (—CH₂CH₂CH₂—), i-propyl(—CHCH₃CH₂—), n-butyl (—CH₂CH₂CH₂CH₂—), s-butyl (—CHCH₃CH₂CH₂—), i-butyl(—C(CH₃)₂CH₂—), n-pentyl (—CH₂CH₂CH₂CH₂CH₂—), s-pentyl(—CHCH₃CH₂CH₂CH₂—) or n-hexyl (—CH₂CH₂CH₂CH₂CH₂CH₂—).

“Alkenyl” includes unsaturated aliphatic groups analogous in length andpossible substitution to the alkyls described above, but that contain atleast one double bond. For example, the term “alkenyl” includes straightchain alkenyl groups (e.g., ethenyl, propenyl, butenyl, pentenyl,hexenyl, heptenyl, octenyl, nonenyl, decenyl), and branched alkenylgroups. In certain embodiments, a straight chain or branched alkenylgroup has six or fewer carbon atoms in its backbone (e.g., C₂-C₆ forstraight chain, C₃-C₆ for branched chain). The term “C₂-C₆” includesalkenyl groups containing two to six carbon atoms. The term “C₃-C₆”includes alkenyl groups containing three to six carbon atoms.

The term “optionally substituted alkenyl” refers to unsubstitutedalkenyl or alkenyl having designated substituents replacing one or morehydrogen atoms on one or more hydrocarbon backbone carbon atoms. Suchsubstituents can include, for example, alkyl, alkenyl, alkynyl, halogen,hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, amino(including alkylamino, dialkylamino, arylamino, diarylamino andalkylarylamino), acylamino (including alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl,alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano,heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.

“Alkynyl” includes unsaturated aliphatic groups analogous in length andpossible substitution to the alkyls described above, but which containat least one triple bond. For example, “alkynyl” includes straight chainalkynyl groups (e.g., ethynyl, propynyl, butynyl, pentynyl, hexynyl,heptynyl, octynyl, nonynyl, decynyl), and branched alkynyl groups. Incertain embodiments, a straight chain or branched alkynyl group has sixor fewer carbon atoms in its backbone (e.g., C₂-C₆ for straight chain,C₃-C₆ for branched chain). The term “C₂-C₆” includes alkynyl groupscontaining two to six carbon atoms. The term “C₃-C₆” includes alkynylgroups containing three to six carbon atoms.

The term “optionally substituted alkynyl” refers to unsubstitutedalkynyl or alkynyl having designated substituents replacing one or morehydrogen atoms on one or more hydrocarbon backbone carbon atoms. Suchsubstituents can include, for example, alkyl, alkenyl, alkynyl, halogen,hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, arylcarbonyl,alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl, dialkylaminocarbonyl,alkylthiocarbonyl, alkoxyl, phosphate, phosphonato, phosphinato, amino(including alkylamino, dialkylamino, arylamino, diarylamino andalkylarylamino), acylamino (including alkylcarbonylamino,arylcarbonylamino, carbamoyl and ureido), amidino, imino, sulfhydryl,alkylthio, arylthio, thiocarboxylate, sulfates, alkylsulfinyl,sulfonato, sulfamoyl, sulfonamido, nitro, trifluoromethyl, cyano, azido,heterocyclyl, alkylaryl, or an aromatic or heteroaromatic moiety.

Other optionally substituted moieties (such as optionally substitutedcycloalkyl, heterocycloalkyl, aryl, or heteroaryl) include both theunsubstituted moieties and the moieties having one or more of thedesignated substituents.

“Aryl” includes groups with aromaticity, including “conjugated,” ormulticyclic systems with at least one aromatic ring and do not containany heteroatom in the ring structure. Examples include phenyl, benzyl,1,2,3,4-tetrahydronaphthalenyl, etc.

“Heteroaryl” groups are aryl groups, as defined above, except havingfrom one to four heteroatoms in the ring structure, and may also bereferred to as “aryl heterocycles” or “heteroaromatics.” As used herein,the term “heteroaryl” is intended to include a stable 5- or 6-memberedmonocyclic or 7-, 8-, 9-, 10-, 11- or 12-membered bicyclic aromaticheterocyclic ring which consists of carbon atoms and one or moreheteroatoms, e.g., 1 or 1-2 or 1-3 or 1-4 or 1-5 or 1-6 heteroatoms, ore.g., 1, 2, 3, 4, 5, or 6 heteroatoms, independently selected from thegroup consisting of nitrogen, oxygen and sulfur. The nitrogen atom maybe substituted or unsubstituted (i.e., N or NR wherein R is H or othersubstituents, as defined). The nitrogen and sulfur heteroatoms mayoptionally be oxidized (i.e., N→O and S(O)_(p), where p=1 or 2). It isto be noted that total number of S and O atoms in the aromaticheterocycle is not more than 1.

Examples of heteroaryl groups include pyrrole, furan, thiophene,thiazole, isothiazole, imidazole, triazole, tetrazole, pyrazole,oxazole, isoxazole, pyridine, pyrazine, pyridazine, pyrimidine, and thelike.

Furthermore, the terms “aryl” and “heteroaryl” include multicyclic aryland heteroaryl groups, e.g., tricyclic, bicyclic, e.g., naphthalene,benzoxazole, benzodioxazole, benzothiazole, benzoimidazole,benzothiophene, methylenedioxyphenyl, quinoline, isoquinoline,naphthrydine, indole, benzofuran, purine, benzofuran, deazapurine,indolizine.

In the case of multicyclic aromatic rings, only one of the rings needsto be aromatic (e.g., 2,3-dihydroindole), although all of the rings maybe aromatic (e.g., quinoline). The second ring can also be fused orbridged.

The aryl or heteroaryl aromatic ring can be substituted at one or morering positions with such substituents as described above, for example,alkyl, alkenyl, alkynyl, halogen, hydroxyl, alkoxy, alkylcarbonyloxy,arylcarbonyloxy, alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate,alkylcarbonyl, alkylaminocarbonyl, aralkylaminocarbonyl,alkenylaminocarbonyl, alkylcarbonyl, arylcarbonyl, aralkylcarbonyl,alkenylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylthiocarbonyl,phosphate, phosphonato, phosphinato, amino (including alkylamino,dialkylamino, arylamino, diarylamino and alkylarylamino), acylamino(including alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido),amidino, imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate,sulfates, alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety. Aryl groups can also be fused or bridged withalicyclic or heterocyclic rings, which are not aromatic so as to form amulticyclic system (e.g., tetralin, methylenedioxyphenyl).

As used herein, “carbocycle” or “carbocyclic ring” is intended toinclude any stable monocyclic, bicyclic or tricyclic ring having thespecified number of carbons, any of which may be saturated, unsaturated,or aromatic. For example, a C₃-C₁₄ carbocycle is intended to include amonocyclic, bicyclic or tricyclic ring having 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13 or 14 carbon atoms. Examples of carbocycles include, but arenot limited to, cyclopropyl, cyclobutyl, cyclobutenyl, cyclopentyl,cyclopentenyl, cyclohexyl, cycloheptenyl, cycloheptyl, cycloheptenyl,adamantyl, cyclooctyl, cyclooctenyl, cyclooctadienyl, fluorenyl, phenyl,naphthyl, indanyl, adamantyl and tetrahydronaphthyl. Bridged rings arealso included in the definition of carbocycle, including, for example,[3.3.0]bicyclooctane, [4.3.0]bicyclononane, [4.4.0]bicyclodecane and[2.2.2]bicyclooctane. A bridged ring occurs when one or more carbonatoms link two non-adjacent carbon atoms. In one embodiment, bridgerings are one or two carbon atoms. It is noted that a bridge alwaysconverts a monocyclic ring into a tricyclic ring. When a ring isbridged, the substituents recited for the ring may also be present onthe bridge. Fused (e.g., naphthyl, tetrahydronaphthyl) and spiro ringsare also included.

As used herein, “heterocycle” includes any ring structure (saturated orpartially unsaturated) which contains at least one ring heteroatom(e.g., N, O or S). Examples of heterocycles include, but are not limitedto, morpholine, pyrrolidine, tetrahydrothiophene, piperidine, piperazineand tetrahydrofuran.

Examples of heterocyclic groups include, but are not limited to,acridinyl, azocinyl, benzimidazolyl, benzofuranyl, benzothiofuranyl,benzothiophenyl, benzoxazolyl, benzoxazolinyl, benzthiazolyl,benztriazolyl, benztetrazolyl, benzisoxazolyl, benzisothiazolyl,benzimidazolinyl, carbazolyl, 4aH-carbazolyl, carbolinyl, chromanyl,chromenyl, cinnolinyl, decahydroquinolinyl, 2H,6H-1,5,2-dithiazinyl,dihydrofuro[2,3-b]tetrahydrofuran, furanyl, furazanyl, imidazolidinyl,imidazolinyl, imidazolyl, 1H-indazolyl, indolenyl, indolinyl,indolizinyl, indolyl, 3H-indolyl, isatinoyl, isobenzofuranyl,isochromanyl, isoindazolyl, isoindolinyl, isoindolyl, isoquinolinyl,isothiazolyl, isoxazolyl, methylenedioxyphenyl, morpholinyl,naphthyridinyl, octahydroisoquinolinyl, oxadiazolyl, 1,2,3-oxadiazolyl,1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl,1,2,4-oxadiazol5(4H)-one, oxazolidinyl, oxazolyl, oxindolyl,pyrimidinyl, phenanthridinyl, phenanthrolinyl, phenazinyl,phenothiazinyl, phenoxathinyl, phenoxazinyl, phthalazinyl, piperazinyl,piperidinyl, piperidonyl, 4-piperidonyl, piperonyl, pteridinyl, purinyl,pyranyl, pyrazinyl, pyrazolidinyl, pyrazolinyl, pyrazolyl, pyridazinyl,pyridooxazole, pyridoimidazole, pyridothiazole, pyridinyl, pyridyl,pyrimidinyl, pyrrolidinyl, pyrrolinyl, 2H-pyrrolyl, pyrrolyl,quinazolinyl, quinolinyl, 4H-quinolizinyl, quinoxalinyl, quinuclidinyl,tetrahydrofuranyl, tetrahydroisoquinolinyl, tetrahydroquinolinyl,tetrazolyl, 6H-1,2,5-thiadiazinyl, 1,2,3-thiadiazolyl,1,2,4-thiadiazolyl, 1,2,5-thiadiazolyl, 1,3,4-thiadiazolyl,thianthrenyl, thiazolyl, thienyl, thienothiazolyl, thienooxazolyl,thienoimidazolyl, thiophenyl, triazinyl, 1,2,3-triazolyl,1,2,4-triazolyl, 1,2,5-triazolyl, 1,3,4-triazolyl and xanthenyl.

The term “substituted,” as used herein, means that any one or morehydrogen atoms on the designated atom is replaced with a selection fromthe indicated groups, provided that the designated atom's normal valencyis not exceeded, and that the substitution results in a stable compound.When a substituent is oxo or keto (i.e., ═O), then 2 hydrogen atoms onthe atom are replaced. Keto substituents are not present on aromaticmoieties. Ring double bonds, as used herein, are double bonds that areformed between two adjacent ring atoms (e.g., C═C, C═N or N═N). “Stablecompound” and “stable structure” are meant to indicate a compound thatis sufficiently robust to survive isolation to a useful degree of purityfrom a reaction mixture, and formulation into an efficacious therapeuticagent.

When a bond to a substituent is shown to cross a bond connecting twoatoms in a ring, then such substituent may be bonded to any atom in thering. When a substituent is listed without indicating the atom via whichsuch substituent is bonded to the rest of the compound of a givenformula, then such substituent may be bonded via any atom in suchformula. Combinations of substituents and/or variables are permissible,but only if such combinations result in stable compounds.

When any variable (e.g., R₁) occurs more than one time in anyconstituent or formula for a compound, its definition at each occurrenceis independent of its definition at every other occurrence. Thus, forexample, if a group is shown to be substituted with 0-2 R₁ moieties,then the group may optionally be substituted with up to two R₁ moietiesand R₁ at each occurrence is selected independently from the definitionof R₁. Also, combinations of substituents and/or variables arepermissible, but only if such combinations result in stable compounds.

The term “hydroxy” or “hydroxyl” includes groups with an —OH or —O⁻.

As used herein, “halo” or “halogen” refers to fluoro, chloro, bromo andiodo. The term “perhalogenated” generally refers to a moiety wherein allhydrogen atoms are replaced by halogen atoms. The term “haloalkyl” or“haloalkoxyl” refers to an alkyl or alkoxyl substituted with one or morehalogen atoms.

The term “carbonyl” includes compounds and moieties which contain acarbon connected with a double bond to an oxygen atom. Examples ofmoieties containing a carbonyl include, but are not limited to,aldehydes, ketones, carboxylic acids, amides, esters, anhydrides, etc.

The term “carboxyl” refers to —COOH or its C₁-C₆ alkyl ester.

“Acyl” includes moieties that contain the acyl radical (R—C(O)—) or acarbonyl group. “Substituted acyl” includes acyl groups where one ormore of the hydrogen atoms are replaced by, for example, alkyl groups,alkynyl groups, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,phosphonato, phosphinato, amino (including alkylamino, dialkylamino,arylamino, diarylamino and alkylarylamino), acylamino (includingalkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino,imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates,alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moiety.

“Aroyl” includes moieties with an aryl or heteroaromatic moiety bound toa carbonyl group. Examples of aroyl groups include phenylcarboxy,naphthyl carboxy, etc.

“Alkoxyalkyl,” “alkylaminoalkyl,” and “thioalkoxyalkyl” include alkylgroups, as described above, wherein oxygen, nitrogen, or sulfur atomsreplace one or more hydrocarbon backbone carbon atoms.

The term “alkoxy” or “alkoxyl” includes substituted and unsubstitutedalkyl, alkenyl and alkynyl groups covalently linked to an oxygen atom.Examples of alkoxy groups or alkoxyl radicals include, but are notlimited to, methoxy, ethoxy, isopropyloxy, propoxy, butoxy and pentoxygroups. Examples of substituted alkoxy groups include halogenated alkoxygroups. The alkoxy groups can be substituted with groups such asalkenyl, alkynyl, halogen, hydroxyl, alkylcarbonyloxy, arylcarbonyloxy,alkoxycarbonyloxy, aryloxycarbonyloxy, carboxylate, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, phosphate,phosphonato, phosphinato, amino (including alkylamino, dialkylamino,arylamino, diarylamino, and alkylarylamino), acylamino (includingalkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido), amidino,imino, sulfhydryl, alkylthio, arylthio, thiocarboxylate, sulfates,alkylsulfinyl, sulfonato, sulfamoyl, sulfonamido, nitro,trifluoromethyl, cyano, azido, heterocyclyl, alkylaryl, or an aromaticor heteroaromatic moieties. Examples of halogen substituted alkoxygroups include, but are not limited to, fluoromethoxy, difluoromethoxy,trifluoromethoxy, chloromethoxy, dichloromethoxy and trichloromethoxy.

The term “ether” or “alkoxy” includes compounds or moieties whichcontain an oxygen bonded to two carbon atoms or heteroatoms. Forexample, the term includes “alkoxyalkyl,” which refers to an alkyl,alkenyl, or alkynyl group covalently bonded to an oxygen atom which iscovalently bonded to an alkyl group.

The term “ester” includes compounds or moieties which contain a carbonor a heteroatom bound to an oxygen atom which is bonded to the carbon ofa carbonyl group. The term “ester” includes alkoxycarboxy groups such asmethoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, butoxycarbonyl,pentoxycarbonyl, etc.

The term “thioalkyl” includes compounds or moieties which contain analkyl group connected with a sulfur atom. The thioalkyl groups can besubstituted with groups such as alkyl, alkenyl, alkynyl, halogen,hydroxyl, alkylcarbonyloxy, arylcarbonyloxy, alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, carboxyacid, alkylcarbonyl,arylcarbonyl, alkoxycarbonyl, aminocarbonyl, alkylaminocarbonyl,dialkylaminocarbonyl, alkylthiocarbonyl, alkoxyl, amino (includingalkylamino, dialkylamino, arylamino, diarylamino and alkylarylamino),acylamino (including alkylcarbonylamino, arylcarbonylamino, carbamoyland ureido), amidino, imino, sulfhydryl, alkylthio, arylthio,thiocarboxylate, sulfates, alkylsulfinyl, sulfonato, sulfamoyl,sulfonamido, nitro, trifluoromethyl, cyano, azido, heterocyclyl,alkylaryl, or an aromatic or heteroaromatic moieties.

The term “thiocarbonyl” or “thiocarboxy” includes compounds and moietieswhich contain a carbon connected with a double bond to a sulfur atom.

The term “thioether” includes moieties which contain a sulfur atombonded to two carbon atoms or heteroatoms. Examples of thioethersinclude, but are not limited to alkthioalkyls, alkthioalkenyls, andalkthioalkynyls. The term “alkthioalkyls” include moieties with analkyl, alkenyl, or alkynyl group bonded to a sulfur atom which is bondedto an alkyl group. Similarly, the term “alkthioalkenyls” refers tomoieties wherein an alkyl, alkenyl or alkynyl group is bonded to asulfur atom which is covalently bonded to an alkenyl group; andalkthioalkynyls” refers to moieties wherein an alkyl, alkenyl or alkynylgroup is bonded to a sulfur atom which is covalently bonded to analkynyl group.

As used herein, “amine” or “amino” refers to unsubstituted orsubstituted —NH₂. “Alkylamino” includes groups of compounds whereinnitrogen of —NH₂ is bound to at least one alkyl group. Examples ofalkylamino groups include benzylamino, methylamino, ethylamino,phenethylamino, etc. “Dialkylamino” includes groups wherein the nitrogenof —NH₂ is bound to at least two additional alkyl groups. Examples ofdialkylamino groups include, but are not limited to, dimethylamino anddiethylamino. “Arylamino” and “diarylamino” include groups wherein thenitrogen is bound to at least one or two aryl groups, respectively.“Aminoaryl” and “aminoaryloxy” refer to aryl and aryloxy substitutedwith amino. “Alkylarylamino,” “alkylaminoaryl” or “arylaminoalkyl”refers to an amino group which is bound to at least one alkyl group andat least one aryl group. “Alkaminoalkyl” refers to an alkyl, alkenyl, oralkynyl group bound to a nitrogen atom which is also bound to an alkylgroup. “Acylamino” includes groups wherein nitrogen is bound to an acylgroup. Examples of acylamino include, but are not limited to,alkylcarbonylamino, arylcarbonylamino, carbamoyl and ureido groups.

The term “amide” or “aminocarboxy” includes compounds or moieties thatcontain a nitrogen atom that is bound to the carbon of a carbonyl or athiocarbonyl group. The term includes “alkaminocarboxy” groups thatinclude alkyl, alkenyl or alkynyl groups bound to an amino group whichis bound to the carbon of a carbonyl or thiocarbonyl group. It alsoincludes “arylaminocarboxy” groups that include aryl or heteroarylmoieties bound to an amino group that is bound to the carbon of acarbonyl or thiocarbonyl group. The terms “alkylaminocarboxy”,“alkenylaminocarboxy”, “alkynylaminocarboxy” and “arylaminocarboxy”include moieties wherein alkyl, alkenyl, alkynyl and aryl moieties,respectively, are bound to a nitrogen atom which is in turn bound to thecarbon of a carbonyl group. Amides can be substituted with substituentssuch as straight chain alkyl, branched alkyl, cycloalkyl, aryl,heteroaryl or heterocycle. Substituents on amide groups may be furthersubstituted.

The term “about”, “approximately”, or “approximate”, when used inconnection with a numerical value, means that a collection or ranger ofvalues is included. For example, “about X” includes a range of valuesthat are ±10%, ±5%, ±2%, ±1%, +0.5%, ±0.2%, or ±0.1% of X, where X is anumerical value. In addition, “about X” may also include a range ofX±0.5, X±0.4, X±0.3, X±0.2, or X±0.1, where X is a numerical value.

Compounds of the present invention that contain nitrogen atoms can beconverted to N-oxides by treatment with an oxidizing agent (e.g.,3-chloroperoxybenzoic acid (mCPBA) and/or hydrogen peroxides) to affordother compounds of the present invention. Thus, all shown and claimednitrogen-containing compounds are considered, when allowed by valencyand structure, to include both the compound as shown and its N-oxidederivative (which can be designated as N→O or N⁺—O⁻). Furthermore, inother instances, the nitrogens in the compounds of the present inventioncan be converted to N-hydroxy or N-alkoxy compounds. For example,N-hydroxy compounds can be prepared by oxidation of the parent amine byan oxidizing agent such as m-CPBA. All shown and claimednitrogen-containing compounds are also considered, when allowed byvalency and structure, to cover both the compound as shown and itsN-hydroxy (i.e., N—OH) and N-alkoxy (i.e., N—OR, wherein R issubstituted or unsubstituted C₁-C₆ alkyl, C₁-C₆ alkenyl, C₁-C₆ alkynyl,3-14-membered carbocycle or 3-14-membered heterocycle) derivatives.

In the present specification, the structural formula of the compoundrepresents a certain isomer for convenience in some cases, but thepresent invention includes all isomers, such as geometrical isomers,optical isomers based on an asymmetrical carbon, stereoisomers,tautomers, and the like. In addition, a crystal polymorphism may bepresent for the compounds represented by the formula. It is noted thatany crystal form, crystal form mixture, or anhydride or hydrate thereofis included in the scope of the present invention. Furthermore,so-called metabolite which is produced by degradation of the presentcompound in vivo is included in the scope of the present invention.

“Isomerism” means compounds that have identical molecular formulae butdiffer in the sequence of bonding of their atoms or in the arrangementof their atoms in space. Isomers that differ in the arrangement of theiratoms in space are termed “stereoisomers.” Stereoisomers that are notmirror images of one another are termed “diastereoisomers,” andstereoisomers that are non-superimposable mirror images of each otherare termed “enantiomers” or sometimes optical isomers. A mixturecontaining equal amounts of individual enantiomeric forms of oppositechirality is termed a “racemic mixture.”

A carbon atom bonded to four nonidentical substituents is termed a“chiral center.”

“Chiral isomer” means a compound with at least one chiral center.Compounds with more than one chiral center may exist either as anindividual diastereomer or as a mixture of diastereomers, termed“diastereomeric mixture.” When one chiral center is present, astereoisomer may be characterized by the absolute configuration (R or S)of that chiral center. Absolute configuration refers to the arrangementin space of the substituents attached to the chiral center. Thesubstituents attached to the chiral center under consideration areranked in accordance with the Sequence Rule of Cahn, Ingold and Prelog.(Cahn et al., Angew. Chem. Inter. Edit. 1966, 5, 385; errata 511; Cahnet al., Angew. Chem. 1966, 78, 413; Cahn and Ingold, J. Chem. Soc. 1951(London), 612; Cahn et al., Experientia 1956, 12, 81; Cahn, J. Chem.Educ. 1964, 41, 116).

“Geometric isomer” means the diastereomers that owe their existence tohindered rotation about double bonds or a cycloalkyl linker (e.g.,1,3-cylcobutyl). These configurations are differentiated in their namesby the prefixes cis and trans, or Z and E, which indicate that thegroups are on the same or opposite side of the double bond in themolecule according to the Cahn-Ingold-Prelog rules.

It is to be understood that the compounds of the present invention maybe depicted as different chiral isomers or geometric isomers. It shouldalso be understood that when compounds have chiral isomeric or geometricisomeric forms, all isomeric forms are intended to be included in thescope of the present invention, and the naming of the compounds does notexclude any isomeric forms.

Furthermore, the structures and other compounds discussed in thisinvention include all atropic isomers thereof. “Atropic isomers” are atype of stereoisomer in which the atoms of two isomers are arrangeddifferently in space. Atropic isomers owe their existence to arestricted rotation caused by hindrance of rotation of large groupsabout a central bond. Such atropic isomers typically exist as a mixture,however as a result of recent advances in chromatography techniques, ithas been possible to separate mixtures of two atropic isomers in selectcases.

“Tautomer” is one of two or more structural isomers that exist inequilibrium and is readily converted from one isomeric form to another.This conversion results in the formal migration of a hydrogen atomaccompanied by a switch of adjacent conjugated double bonds. Tautomersexist as a mixture of a tautomeric set in solution. In solutions wheretautomerization is possible, a chemical equilibrium of the tautomerswill be reached. The exact ratio of the tautomers depends on severalfactors, including temperature, solvent and pH. The concept of tautomersthat are interconvertable by tautomerizations is called tautomerism.

Of the various types of tautomerism that are possible, two are commonlyobserved. In keto-enol tautomerism a simultaneous shift of electrons anda hydrogen atom occurs. Ring-chain tautomerism arises as a result of thealdehyde group (—CHO) in a sugar chain molecule reacting with one of thehydroxy groups (—OH) in the same molecule to give it a cyclic(ring-shaped) form as exhibited by glucose.

Common tautomeric pairs are: ketone-enol, amide-nitrile, lactam-lactim,amide-imidic acid tautomerism in heterocyclic rings (e.g., innucleobases such as guanine, thymine and cytosine), amine-enamine andenamine-enamine. Benzimidazoles also exhibit tautomerism, when thebenzimidazole contains one or more substituents in the 4, 5, 6 or 7positions, the possibility of different isomers arises. For example,2,5-dimethyl-1H-benzo[d]imidazole can exist in equilibrium with itsisomer 2,6-dimethyl-1H-benzo[d]imidazole via tautomerization.

Another example of tautomerism is shown below.

It is to be understood that the compounds of the present invention maybe depicted as different tautomers. It should also be understood thatwhen compounds have tautomeric forms, all tautomeric forms are intendedto be included in the scope of the present invention, and the naming ofthe compounds does not exclude any tautomer form.

The term “crystal polymorphs”, “polymorphs” or “crystalline forms” meanscrystal structures in which a compound (or a salt or solvate thereof)can crystallize in different crystal packing arrangements, all of whichhave the same elemental composition. Different crystal forms usuallyhave different XRPD patterns, infrared spectral, melting points, densityhardness, crystal shape, optical and electrical properties, stabilityand solubility. Recrystallization solvent, rate of crystallization,storage temperature, and other factors may cause one crystal form todominate. Crystal polymorphs of the compounds can be prepared bycrystallization under different conditions.

Compounds of the invention may be crystalline, semi-crystalline,non-crystalline, amorphous, mesomorphous, etc.

The compounds of Formula (I) and other compounds of the inventioninclude the compounds themselves, as well as their N-oxides, salts,their solvates, and their prodrugs, if applicable. A salt, for example,can be formed between an anion and a positively charged group (e.g.,amino) on a substituted purine or 7-deazapurine compound. Suitableanions include chloride, bromide, iodide, sulfate, bisulfate, sulfamate,nitrate, phosphate, citrate, methanesulfonate, trifluoroacetate,glutamate, glucuronate, glutarate, malate, maleate, succinate, fumarate,tartrate, tosylate, salicylate, lactate, naphthalenesulfonate, andacetate. Likewise, a salt can also be formed between a cation and anegatively charged group (e.g., carboxylate) on a substituted purine or7-deazapurine compound. Suitable cations include sodium ion, potassiumion, magnesium ion, calcium ion, and an ammonium cation such astetramethylammonium ion. The substituted purine or 7-deazapurinecompounds also include those salts containing quaternary nitrogen atoms.Examples of prodrugs include esters and other pharmaceuticallyacceptable derivatives, which, upon administration to a subject, arecapable of providing active substituted purine or 7-deazapurinecompounds.

Additionally, the compounds or crystalline forms of the presentinvention, for example, the salts of the compounds or crystalline forms,can exist in either hydrated or unhydrated (the anhydrous) form or assolvates with other solvent molecules. Nonlimiting examples of hydratesinclude hemihydrates, monohydrates, dihydrates, trihydrates, etc.Nonlimiting examples of solvates include ethanol solvates, acetonesolvates, etc.

“Solvate” means solvent addition forms that contain eitherstoichiometric or non stoichiometric amounts of solvent. For example, asolvate can have one or more solvent molecule per compound molecule,e.g., 1, 2, 3, 4, or more solvent molecules. Some compounds have atendency to trap a fixed molar ratio of solvent molecules in thecrystalline solid state, thus forming a solvate. If the solvent is waterthe solvate formed is a hydrate; and if the solvent is alcohol, thesolvate formed is an alcoholate. Hydrates are formed by the combinationof one or more molecules of water with one molecule of the substance inwhich the water retains its molecular state as H₂O. A hemihydrate isformed by the combination of one molecule of water with more than onemolecule of the substance in which the water retains its molecular stateas H₂O.

As used herein, the term “analog” refers to a chemical compound that isstructurally similar to another but differs slightly in composition (asin the replacement of one atom by an atom of a different element or inthe presence of a particular functional group, or the replacement of onefunctional group by another functional group). Thus, an analog is acompound that is similar or comparable in function and appearance, butnot in structure or origin to the reference compound.

As defined herein, the term “derivative” refers to compounds that have acommon core structure, and are substituted with various groups asdescribed herein. For example, all of the compounds represented byFormula (I) are substituted purine compounds or substituted7-deazapurine compounds, and have Formula (I) as a common core.

The term “bioisostere” refers to a compound resulting from the exchangeof an atom or of a group of atoms with another, broadly similar, atom orgroup of atoms. The objective of a bioisosteric replacement is to createa new compound with similar biological properties to the parentcompound. The bioisosteric replacement may be physicochemically ortopologically based. Examples of carboxylic acid bioisosteres include,but are not limited to, acyl sulfonimides, tetrazoles, sulfonates andphosphonates. See, e.g., Patani and LaVoie, Chem. Rev. 96, 3147-3176,1996.

The present invention is intended to include all isotopes of atomsoccurring in the present compounds. Isotopes include those atoms havingthe same atomic number but different mass numbers. By way of generalexample and without limitation, isotopes of hydrogen include tritium anddeuterium, and isotopes of carbon include C-13 and C-14.

The present invention provides methods of treating or preventing cancer.The present invention provides methods of treating cancer. The presentinvention also provides methods of preventing cancer. The methodincludes administering to a subject in need thereof a therapeuticallyeffective amount of the compound of the invention. The cancer can be ahematological cancer. Preferably, the cancer is leukemia. Morepreferably, the cancer is acute myeloid leukemia, acute lymphocyticleukemia or mixed lineage leukemia.

The present invention provides methods of treating or preventing adisease or disorder mediated by translocation of a gene on chromosome11q23. The present invention provides methods of treating a disease ordisorder mediated by translocation of a gene on chromosome 11q23. Thepresent invention also provides methods of preventing a disease ordisorder mediated by translocation of a gene on chromosome 11q23. Themethod includes administering to a subject in need thereof atherapeutically effective amount of the compound or crystalline form ofthe invention.

The present invention provides methods of treating or preventing adisease or disorder in which DOT1-mediated protein methylation plays apart or a disease or disorder mediated by DOT1-mediated proteinmethylation. The present invention provides methods of treating adisease or disorder in which DOT1-mediated protein methylation plays apart or a disease or disorder mediated by DOT1-mediated proteinmethylation. The present invention also provides methods of preventing adisease or disorder in which DOT1-mediated protein methylation plays apart or a disease or disorder mediated by DOT1-mediated proteinmethylation. The method includes administering to a subject in needthereof a therapeutically effective amount of the compound orcrystalline form of the invention.

The present invention provides methods of inhibiting DOT1L activity in acell. The method includes contacting the cell with an effective amountof one or more of the compound or crystalline form of the invention.

Still another aspect of the invention relates to a method of reducingthe level of Histone H3 Lysine residue 79 (H3-K79) methylation in acell. The method includes contacting a cell with a compound of thepresent invention. Such method can be used to ameliorate any conditionwhich is caused by or potentiated by the activity of DOT1 through H3-K79methylation.

The present invention relates to use of the compounds disclosed hereinin preparation of a medicament for treating or preventing cancer. Theuse includes a compound or crystalline form of the invention foradministration to a subject in need thereof in a therapeuticallyeffective amount. The cancer can be a hematological cancer. Preferably,the cancer is leukemia. More preferably, the cancer is acute myeloidleukemia, acute lymphocytic leukemia or mixed lineage leukemia.

The present invention provides use of the compounds disclosed herein inpreparation of a medicament for treating or preventing a disease ordisorder mediated by translocation of a gene on chromosome 11q23. Theuse includes a compound or crystalline form of the invention foradministration to a subject in need thereof in a therapeuticallyeffective amount.

The present invention provides use of the compounds disclosed herein inpreparation of a medicament for treating or preventing a disease ordisorder in which DOT1-mediated protein methylation plays a part or adisease or disorder mediated by DOT1-mediated protein methylation. Theuse includes a compound or crystalline form of the invention foradministration to a subject in need thereof in a therapeuticallyeffective amount.

The present invention provides use of the compounds disclosed herein forinhibiting DOT1L activity in a cell. The use includes contacting thecell with an effective amount of one or more of the compound orcrystalline form of the invention.

Still another aspect of the invention relates to a use of the compoundsdisclosed herein for reducing the level of Histone H3 Lysine residue 79(H3-K79) methylation in a cell. The use includes contacting a cell witha compound of the present invention. Such use can ameliorate anycondition which is caused by or potentiated by the activity of DOT1through H3-K79 methylation.

In the formula presented herein, the variables can be selected from therespective groups of chemical moieties later defined in the detaileddescription.

In addition, the invention provides methods of synthesizing theforegoing compounds. Following synthesis, a therapeutically effectiveamount of one or more of the compounds can be formulated with apharmaceutically acceptable carrier for administration to a mammal,particularly humans, for use in modulating an epigenetic enzyme. Incertain embodiments, the compounds of the present invention are usefulfor treating, preventing, or reducing the risk of cancer or for themanufacture of a medicament for treating, preventing, or reducing therisk of cancer. Accordingly, the compounds or the formulations can beadministered, for example, via oral, parenteral, otic, ophthalmic,nasal, or topical routes, to provide an effective amount of the compoundto the mammal.

Mixed lineage leukemia (MLL) is a genetically distinct form of acuteleukemia that constitutes over 70% of infant leukemias and approximately10% of adult acute myeloid leukemias (AML) (Hess, J. L. (2004), TrendsMol Med 10, 500-507; Krivtsov, A. V., and Armstrong, S. A. (2007), NatRev Cancer 7, 823-833). MLL represents a particularly aggressive form ofleukemia and patients with this disease generally have poor prognoses;these patients often suffer from early relapse after treatment withcurrent chemotherapies. There is thus a great and present need for newtreatment modalities for patients suffering with MLL.

A universal hallmark of MLL disease is a chromosomal translocationaffecting the MLL gene on chromosome 11q23 (Hess, 2004; Krivtsov andArmstrong, 2007). Normally, the MLL gene encodes for a SET-domainhistone methyltransferase that catalyzes the methylation of lysine 4 ofhistone H3 (H3K4) at specific gene loci (Milne et al. (2002) Mol Cell10, 1107-1117; Nakamura et al. (2002), Mol Cell 10, 1119-1128). Genelocalization is conferred by specific interactions with recognitionelements within MLL, external to the SET-domain (Ayton et al. (2004) MolCell Biol 24, 10470-10478; Slany et al., (1998) Mol Cell Biol 18,122-129; Zeleznik-Le et al. (1994) Proc Natl Acad Sci USA 91,10610-10614). In the disease-linked translocations, the catalyticSET-domain is lost and the remaining MLL protein is fused to a varietyof partners, including members of the AF and ENL family of proteins suchas AF4, AF9, AF10 and ENL (Hess, 2004; Krivtsov and Armstrong, 2007;Slany (2009) Haematologica 94, 984-993). These fusion partners arecapable of interacting directly, or indirectly, with another histonemethyltransferase, DOT1L (Bitoun et al. (2007) Hum Mol Genet 16, 92-106;Mohan et al. (2010) Genes Dev. 24, 574-589; Mueller et al. (2007) Blood110, 4445-4454; Mueller et al. (2009) PLoS Biol 7, e1000249; Okada etal. (2005) Cell 121, 167-178; Park et al. (2010) Protein J 29, 213-223;Yokoyama et al. (2010) Cancer Cell 17, 198-212; Zhang et al. (2006) JBiol Chem 281, 18059-18068). As a result, translocation products retaingene-specific recognition elements within the remainder of the MLLprotein, but also gain the ability to recruit DOT1L, to these locations(Monroe et al. (2010) Exp Hematol. 2010 Sep. 18. [Epub ahead of print]Pubmed PMID: 20854876; Mueller et al., 2007; Mueller et al., 2009; Okadaet al., 2005). DOT1L catalyzes the methylation of H3K79, a chromatinmodification associated with actively transcribed genes (Feng et al.(2002) Curr Biol 12, 1052-1058; Steger et al. (2008) Mol Cell Biol 28,2825-2839). The ectopic H3K79 methylation that results from MLL fusionprotein recruitment of DOT1L leads to enhanced expression ofleukemogenic genes, including HOXA9 and MEIS1 (Guenther et al. (2008)Genes & Development 22, 3403-3408; Krivtsov et al. (2008) Nat Rev Cancer7, 823-833; Milne et al. (2005) Cancer Res 65, 11367-11374; Monroe etal., 2010; Mueller et al., 2009; Okada et al., 2005; Thiel et al. (2010)Cancer Cell 17, 148-159). Hence, while DOT1L is not genetically alteredin the disease per se, its mislocated enzymatic activity is a directconsequence of the chromosomal translocation affecting MLL patients;thus, DOT1L has been proposed to be a catalytic driver of leukemogenesisin this disease (Krivtsov et al., 2008; Monroe et al., 2010; Okada etal., 2005; Yokoyama et al. (2010) Cancer Cell 17, 198-212). Furthersupport for a pathogenic role of DOT1L in MLL comes from studies inmodel systems that demonstrate a requirement for DOT1L in propagatingthe transforming activity of MLL fusion proteins (Mueller et al., 2007;Okada et al., 2005).

Evidence indicates that the enzymatic activity of DOT1L is critical topathogenesis in MLL and inhibition of DOT1L may provide a pharmacologicbasis for therapeutic intervention in this disease. Compound treatmentresults in selective, concentration-dependent killing of leukemia cellsbearing the MLL-translocation without effect on non-MLL transformedcells. Gene expression analysis of inhibitor treated cells showsdownregulation of genes aberrantly over expressed in MLL-rearrangedleukemias and similarities with gene expression changes caused bygenetic knockout of the Dot1L gene in a mouse model of MLL-AF9 leukemia.

The present invention provides methods for the treatment of a cellproliferative disorder in a subject in need thereof by administering toa subject in need of such treatment, a therapeutically effective amountof a compound of the present invention, or a pharmaceutically acceptablesalt, prodrug, metabolite, crystalline form or solvate thereof. The cellproliferative disorder can be cancer or a precancerous condition. Thepresent invention further provides the use of a compound of the presentinvention, or a pharmaceutically acceptable salt, prodrug, metabolite,crystalline form or solvate thereof, for the preparation of a medicamentuseful for the treatment of a cell proliferative disorder.

The present invention provides methods for the treatment ofhematological cancer or hematologic tumors in a subject in need thereofby administering to a subject in need of such treatment, atherapeutically effective amount of a compound of the present invention,or a pharmaceutically acceptable salt, prodrug, metabolite, crystallineform or solvate thereof. The present invention further provides the useof a compound of the present invention, or a pharmaceutically acceptablesalt, prodrug, metabolite, crystalline form or solvate thereof, for thepreparation of a medicament useful for the treatment of hematologicalcancer or hematologic tumors.

The present invention provides methods for the treatment of leukemia ina subject in need thereof by administering to a subject in need of suchtreatment, a therapeutically effective amount of a compound of thepresent invention, or a pharmaceutically acceptable salt, prodrug,metabolite, crystalline form or solvate thereof. The leukemia can beacute or chronic leukemia. Preferably, the leukemia is acute myeloidleukemia, acute lymphocytic leukemia or mixed lineage leukemia. Thepresent invention further provides the use of a compound of the presentinvention, or a pharmaceutically acceptable salt, prodrug, metabolite,crystalline form or solvate thereof, for the preparation of a medicamentuseful for the treatment of leukemia.

The present invention provides methods for the treatment of a disease ordisorder mediated by translocation of a gene on chromosome 11q23 in asubject in need thereof by administering to a subject in need of suchtreatment, a therapeutically effective amount of a compound of thepresent invention, or a pharmaceutically acceptable salt, prodrug,metabolite, crystalline form or solvate thereof. The gene can be the MLLgene. The present invention further provides the use of a compound ofthe present invention, or a pharmaceutically acceptable salt, prodrug,metabolite, crystalline form or solvate thereof, for the preparation ofa medicament useful for the treatment of a disease or disorder mediatedby translocation of a gene on chromosome 11q23.

The present invention provides methods for the treatment of a disease ordisorder mediated by DOT1 (e.g., DOT1L)-mediated protein methylation ina subject in need thereof by administering to a subject in need of suchtreatment, a therapeutically effective amount of a compound of thepresent invention, or a pharmaceutically acceptable salt, prodrug,metabolite, crystalline form or solvate thereof. The present inventionfurther provides the use of a compound of the present invention, or apharmaceutically acceptable salt, prodrug, metabolite, crystalline formor solvate thereof, for the preparation of a medicament useful for thetreatment of a disease or disorder mediated by DOT1L-mediated proteinmethylation.

The present invention provides methods for the treatment of a disorderthe course of which is influenced by modulating the methylation statusof histones or other proteins, wherein said methylation status ismediated at least in part by the activity of DOT1L. Modulation of themethylation status of histones can in turn influence the level ofexpression of target genes activated by methylation, and/or target genessuppressed by methylation. The method includes administering to asubject in need of such treatment, a therapeutically effective amount ofa compound of the present invention, or a pharmaceutically acceptablesalt, prodrug, metabolite, crystalline form, solvate, or stereoisomerorthereof.

The disorder in which DOT1L-mediated protein methylation plays a partcan be cancer or a precancerous condition or a neurological disease. Thepresent invention further provides the use of a compound of the presentinvention, or a pharmaceutically acceptable salt, prodrug, metabolite,crystalline form or solvate thereof, for the preparation of a medicamentuseful for the treatment of cancer or a neurological disease.

The present invention also provides methods of protecting against adisorder in which DOT1L-mediated protein methylation plays a part in asubject in need thereof by administering a therapeutically effectiveamount of compound of the present invention, or a pharmaceuticallyacceptable salt, prodrug, metabolite, crystalline form or solvatethereof, to a subject in need of such treatment. The disorder can becancer or a neurological disease. The present invention also providesthe use of compound of the present invention, or a pharmaceuticallyacceptable salt, prodrug, metabolite, crystalline form, solvate, orstereoisomeror thereof, for the preparation of a medicament useful forthe prevention of a cell proliferative disorder.

The compounds of this invention can be used to modulate protein (e.g.,histone) methylation, e.g., to modulate histone methyltransferase orhistone demethylase enzyme activity. Histone methylation has beenreported to be involved in aberrant expression of certain genes incancers, and in silencing of neuronal genes in non-neuronal cells. Thecompounds described herein can be used to treat these diseases, i.e., todecreases methylation or restores methylation to roughly its level incounterpart normal cells.

In general, compounds that are methylation modulators can be used formodulating cell proliferation, generally. For example, in some casesexcessive proliferation may be reduced with agents that decreasemethylation, whereas insufficient proliferation may be stimulated withagents that increase methylation. Accordingly, diseases that may betreated by the compounds of the invention include hyperproliferativediseases, such as benign cell growth and malignant cell growth.

As used herein, a “subject in need thereof” is a subject having a cellproliferative disorder, or a subject having an increased risk ofdeveloping a cell proliferative disorder relative to the population atlarge. The subject can have cancer or pre-cancer. Preferably, a subjectin need thereof has cancer. More preferably, a hematologic cancer orleukemia. A “subject” includes a mammal. The mammal can be e.g., anymammal, e.g., a human, primate, bird, mouse, rat, fowl, dog, cat, cow,horse, goat, camel, sheep or a pig. Preferably, the mammal is a human.

As used herein, the term “cell proliferative disorder” refers toconditions in which unregulated or abnormal growth, or both, of cellscan lead to the development of an unwanted condition or disease, whichmay or may not be cancerous. Exemplary cell proliferative disorders ofthe invention encompass a variety of conditions wherein cell division isderegulated. Exemplary cell proliferative disorder include, but are notlimited to, neoplasms, benign tumors, malignant tumors, pre-cancerousconditions, in situ tumors, encapsulated tumors, metastatic tumors,liquid tumors, solid tumors, immunological tumors, hematological tumors,cancers, carcinomas, leukemias, lymphomas, sarcomas, and rapidlydividing cells. The term “rapidly dividing cell” as used herein isdefined as any cell that divides at a rate that exceeds or is greaterthan what is expected or observed among neighboring or juxtaposed cellswithin the same tissue. A cell proliferative disorder includes aprecancer or a precancerous condition. A cell proliferative disorderincludes cancer. Preferably, the methods provided herein are used totreat or alleviate a symptom of cancer. The term “cancer” includes solidtumors, as well as, hematologic tumors and/or malignancies. A “precancercell” or “precancerous cell” is a cell manifesting a cell proliferativedisorder that is a precancer or a precancerous condition. A “cancercell” or “cancerous cell” is a cell manifesting a cell proliferativedisorder that is a cancer. Any reproducible means of measurement may beused to identify cancer cells or precancerous cells. Cancer cells orprecancerous cells can be identified by histological typing or gradingof a tissue sample (e.g., a biopsy sample). Cancer cells or precancerouscells can be identified through the use of appropriate molecularmarkers.

Exemplary non-cancerous conditions or disorders include, but are notlimited to, rheumatoid arthritis; inflammation; autoimmune disease;lymphoproliferative conditions; acromegaly; rheumatoid spondylitis;osteoarthritis; gout, other arthritic conditions; sepsis; septic shock;endotoxic shock; gram-negative sepsis; toxic shock syndrome; asthma;adult respiratory distress syndrome; chronic obstructive pulmonarydisease; chronic pulmonary inflammation; inflammatory bowel disease;Crohn's disease; psoriasis; eczema; ulcerative colitis; pancreaticfibrosis; hepatic fibrosis; acute and chronic renal disease; irritablebowel syndrome; pyresis; restenosis; cerebral malaria; stroke andischemic injury; neural trauma; Alzheimer's disease; Huntington'sdisease; Parkinson's disease; acute and chronic pain; allergic rhinitis;allergic conjunctivitis; chronic heart failure; acute coronary syndrome;cachexia; malaria; leprosy; leishmaniasis; Lyme disease; Reiter'ssyndrome; acute synovitis; muscle degeneration, bursitis; tendonitis;tenosynovitis; herniated, ruptures, or prolapsed intervertebral disksyndrome; osteopetrosis; thrombosis; restenosis; silicosis; pulmonarysarcosis; bone resorption diseases, such as osteoporosis;graft-versus-host reaction; Multiple Sclerosis; lupus; fibromyalgia;AIDS and other viral diseases such as Herpes Zoster, Herpes Simplex I orII, influenza virus and cytomegalovirus; and diabetes mellitus.

Exemplary cancers include, but are not limited to, adrenocorticalcarcinoma, AIDS-related cancers, AIDS-related lymphoma, anal cancer,anorectal cancer, cancer of the anal canal, appendix cancer, childhoodcerebellar astrocytoma, childhood cerebral astrocytoma, basal cellcarcinoma, skin cancer (non-melanoma), biliary cancer, extrahepatic bileduct cancer, intrahepatic bile duct cancer, bladder cancer, uringarybladder cancer, bone and joint cancer, osteosarcoma and malignantfibrous histiocytoma, brain cancer, brain tumor, brain stem glioma,cerebellar astrocytoma, cerebral astrocytoma/malignant glioma,ependymoma, medulloblastoma, supratentorial primitive neuroectodeimaltumors, visual pathway and hypothalamic glioma, breast cancer, bronchialadenomas/carcinoids, carcinoid tumor, gastrointestinal, nervous systemcancer, nervous system lymphoma, central nervous system cancer, centralnervous system lymphoma, cervical cancer, childhood cancers, chroniclymphocytic leukemia, chronic myelogenous leukemia, chronicmyeloproliferative disorders, colon cancer, colorectal cancer, cutaneousT-cell lymphoma, lymphoid neoplasm, mycosis fungoides, Seziary Syndrome,endometrial cancer, esophageal cancer, extracranial germ cell tumor,extragonadal germ cell tumor, extrahepatic bile duct cancer, eye cancer,intraocular melanoma, retinoblastoma, gallbladder cancer, gastric(stomach) cancer, gastrointestinal carcinoid tumor, gastrointestinalstromal tumor (GIST), germ cell tumor, ovarian germ cell tumor,gestational trophoblastic tumor glioma, head and neck cancer,hepatocellular (liver) cancer, Hodgkin lymphoma, hypopharyngeal cancer,intraocular melanoma, ocular cancer, islet cell tumors (endocrinepancreas), Kaposi Sarcoma, kidney cancer, renal cancer, kidney cancer,laryngeal cancer, acute lymphoblastic leukemia, acute lymphocyticleukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronicmyelogenous leukemia, hairy cell leukemia, lip and oral cavity cancer,liver cancer, lung cancer, non-small cell lung cancer, small cell lungcancer, AIDS-related lymphoma, non-Hodgkin lymphoma, primary centralnervous system lymphoma, Waldenstram macroglobulinemia, medulloblastoma,melanoma, intraocular (eye) melanoma, merkel cell carcinoma,mesothelioma malignant, mesothelioma, metastatic squamous neck cancer,mouth cancer, cancer of the tongue, multiple endocrine neoplasiasyndrome, mycosis fungoides, myelodysplastic syndromes,myelodysplastic/myeloproliferative diseases, chronic myelogenousleukemia, acute myeloid leukemia, multiple myeloma, chronicmyeloproliferative disorders, nasopharyngeal cancer, neuroblastoma, oralcancer, oral cavity cancer, oropharyngeal cancer, ovarian cancer,ovarian epithelial cancer, ovarian low malignant potential tumor,pancreatic cancer, islet cell pancreatic cancer, paranasal sinus andnasal cavity cancer, parathyroid cancer, penile cancer, pharyngealcancer, pheochromocytoma, pineoblastoma and supratentorial primitiveneuroectodermal tumors, pituitary tumor, plasma cell neoplasm/multiplemyeloma, pleuropulmonary blastoma, prostate cancer, rectal cancer, renalpelvis and ureter, transitional cell cancer, retinoblastoma,rhabdomyosarcoma, salivary gland cancer, ewing family of sarcoma tumors,Kaposi Sarcoma, soft tissue sarcoma, uterine cancer, uterine sarcoma,skin cancer (non-melanoma), skin cancer (melanoma), merkel cell skincarcinoma, small intestine cancer, soft tissue sarcoma, squamous cellcarcinoma, stomach (gastric) cancer, supratentorial primitiveneuroectodermal tumors, testicular cancer, throat cancer, thymoma,thymoma and thymic carcinoma, thyroid cancer, transitional cell cancerof the renal pelvis and ureter and other urinary organs, gestationaltrophoblastic tumor, urethral cancer, endometrial uterine cancer,uterine sarcoma, uterine corpus cancer, vaginal cancer, vulvar cancer,and Wilm's Tumor.

A “cell proliferative disorder of the hematologic system” is a cellproliferative disorder involving cells of the hematologic system. A cellproliferative disorder of the hematologic system can include lymphoma,leukemia, myeloid neoplasms, mast cell neoplasms, myelodysplasia, benignmonoclonal gammopathy, lymphomatoid granulomatosis, lymphomatoidpapulosis, polycythemia vera, chronic myelocytic leukemia, agnogenicmyeloid metaplasia, and essential thrombocythemia. A cell proliferativedisorder of the hematologic system can include hyperplasia, dysplasia,and metaplasia of cells of the hematologic system. Preferably,compositions of the present invention may be used to treat a cancerselected from the group consisting of a hematologic cancer of thepresent invention or a hematologic cell proliferative disorder of thepresent invention. A hematologic cancer of the present invention caninclude multiple myeloma, lymphoma (including Hodgkin's lymphoma,non-Hodgkin's lymphoma, childhood lymphomas, and lymphomas oflymphocytic and cutaneous origin), leukemia (including childhoodleukemia, hairy-cell leukemia, acute lymphocytic leukemia, acutemyelocytic leukemia, chronic lymphocytic leukemia, chronic myelocyticleukemia, chronic myelogenous leukemia, and mast cell leukemia), myeloidneoplasms and mast cell neoplasms.

A “cell proliferative disorder of the lung” is a cell proliferativedisorder involving cells of the lung. Cell proliferative disorders ofthe lung can include all forms of cell proliferative disorders affectinglung cells. Cell proliferative disorders of the lung can include lungcancer, a precancer or precancerous condition of the lung, benigngrowths or lesions of the lung, and malignant growths or lesions of thelung, and metastatic lesions in tissue and organs in the body other thanthe lung. Preferably, compositions of the present invention may be usedto treat lung cancer or cell proliferative disorders of the lung. Lungcancer can include all forms of cancer of the lung. Lung cancer caninclude malignant lung neoplasms, carcinoma in situ, typical carcinoidtumors, and atypical carcinoid tumors. Lung cancer can include smallcell lung cancer (“SCLC”), non-small cell lung cancer (“NSCLC”),squamous cell carcinoma, adenocarcinoma, small cell carcinoma, largecell carcinoma, adenosquamous cell carcinoma, and mesothelioma. Lungcancer can include “scar carcinoma,” bronchioalveolar carcinoma, giantcell carcinoma, spindle cell carcinoma, and large cell neuroendocrinecarcinoma. Lung cancer can include lung neoplasms having histologic andultrastructual heterogeneity (e.g., mixed cell types).

Cell proliferative disorders of the lung can include all forms of cellproliferative disorders affecting lung cells. Cell proliferativedisorders of the lung can include lung cancer, precancerous conditionsof the lung. Cell proliferative disorders of the lung can includehyperplasia, metaplasia, and dysplasia of the lung. Cell proliferativedisorders of the lung can include asbestos-induced hyperplasia, squamousmetaplasia, and benign reactive mesothelial metaplasia. Cellproliferative disorders of the lung can include replacement of columnarepithelium with stratified squamous epithelium, and mucosal dysplasia.Individuals exposed to inhaled injurious environmental agents such ascigarette smoke and asbestos may be at increased risk for developingcell proliferative disorders of the lung. Prior lung diseases that maypredispose individuals to development of cell proliferative disorders ofthe lung can include chronic interstitial lung disease, necrotizingpulmonary disease, scleroderma, rheumatoid disease, sarcoidosis,interstitial pneumonitis, tuberculosis, repeated pneumonias, idiopathicpulmonary fibrosis, granulomata, asbestosis, fibrosing alveolitis, andHodgkin's disease.

A “cell proliferative disorder of the colon” is a cell proliferativedisorder involving cells of the colon. Preferably, the cellproliferative disorder of the colon is colon cancer. Preferably,compositions of the present invention may be used to treat colon canceror cell proliferative disorders of the colon. Colon cancer can includeall forms of cancer of the colon. Colon cancer can include sporadic andhereditary colon cancers. Colon cancer can include malignant colonneoplasms, carcinoma in situ, typical carcinoid tumors, and atypicalcarcinoid tumors. Colon cancer can include adenocarcinoma, squamous cellcarcinoma, and adenosquamous cell carcinoma. Colon cancer can beassociated with a hereditary syndrome selected from the group consistingof hereditary nonpolyposis colorectal cancer, familial adenomatouspolyposis, Gardner's syndrome, Peutz-Jeghers syndrome, Turcot's syndromeand juvenile polyposis. Colon cancer can be caused by a hereditarysyndrome selected from the group consisting of hereditary nonpolyposiscolorectal cancer, familial adenomatous polyposis, Gardner's syndrome,Peutz-Jeghers syndrome, Turcot's syndrome and juvenile polyposis.

Cell proliferative disorders of the colon can include all forms of cellproliferative disorders affecting colon cells. Cell proliferativedisorders of the colon can include colon cancer, precancerous conditionsof the colon, adenomatous polyps of the colon and metachronous lesionsof the colon. A cell proliferative disorder of the colon can includeadenoma. Cell proliferative disorders of the colon can be characterizedby hyperplasia, metaplasia, and dysplasia of the colon. Prior colondiseases that may predispose individuals to development of cellproliferative disorders of the colon can include prior colon cancer.Current disease that may predispose individuals to development of cellproliferative disorders of the colon can include Crohn's disease andulcerative colitis. A cell proliferative disorder of the colon can beassociated with a mutation in a gene selected from the group consistingof p53, ras, FAP and DCC. An individual can have an elevated risk ofdeveloping a cell proliferative disorder of the colon due to thepresence of a mutation in a gene selected from the group consisting ofp53, ras, FAP and DCC.

A “cell proliferative disorder of the pancreas” is a cell proliferativedisorder involving cells of the pancreas. Cell proliferative disordersof the pancreas can include all forms of cell proliferative disordersaffecting pancreatic cells. Cell proliferative disorders of the pancreascan include pancreas cancer, a precancer or precancerous condition ofthe pancreas, hyperplasia of the pancreas, and dysaplasia of thepancreas, benign growths or lesions of the pancreas, and malignantgrowths or lesions of the pancreas, and metastatic lesions in tissue andorgans in the body other than the pancreas. Pancreatic cancer includesall forms of cancer of the pancreas. Pancreatic cancer can includeductal adenocarcinoma, adenosquamous carcinoma, pleomorphic giant cellcarcinoma, mucinous adenocarcinoma, osteoclast-like giant cellcarcinoma, mucinous cystadenocarcinoma, acinar carcinoma, unclassifiedlarge cell carcinoma, small cell carcinoma, pancreatoblastoma, papillaryneoplasm, mucinous cystadenoma, papillary cystic neoplasm, and serouscystadenoma. Pancreatic cancer can also include pancreatic neoplasmshaving histologic and ultrastructual heterogeneity (e.g., mixed celltypes).

A “cell proliferative disorder of the prostate” is a cell proliferativedisorder involving cells of the prostate. Cell proliferative disordersof the prostate can include all forms of cell proliferative disordersaffecting prostate cells. Cell proliferative disorders of the prostatecan include prostate cancer, a precancer or precancerous condition ofthe prostate, benign growths or lesions of the prostate, and malignantgrowths or lesions of the prostate, and metastatic lesions in tissue andorgans in the body other than the prostate. Cell proliferative disordersof the prostate can include hyperplasia, metaplasia, and dysplasia ofthe prostate.

A “cell proliferative disorder of the skin” is a cell proliferativedisorder involving cells of the skin. Cell proliferative disorders ofthe skin can include all forms of cell proliferative disorders affectingskin cells. Cell proliferative disorders of the skin can include aprecancer or precancerous condition of the skin, benign growths orlesions of the skin, melanoma, malignant melanoma and other malignantgrowths or lesions of the skin, and metastatic lesions in tissue andorgans in the body other than the skin. Cell proliferative disorders ofthe skin can include hyperplasia, metaplasia, and dysplasia of the skin.

A “cell proliferative disorder of the ovary” is a cell proliferativedisorder involving cells of the ovary. Cell proliferative disorders ofthe ovary can include all forms of cell proliferative disordersaffecting cells of the ovary. Cell proliferative disorders of the ovarycan include a precancer or precancerous condition of the ovary, benigngrowths or lesions of the ovary, ovarian cancer, malignant growths orlesions of the ovary, and metastatic lesions in tissue and organs in thebody other than the ovary. Cell proliferative disorders of the skin caninclude hyperplasia, metaplasia, and dysplasia of cells of the ovary.

A “cell proliferative disorder of the breast” is a cell proliferativedisorder involving cells of the breast. Cell proliferative disorders ofthe breast can include all forms of cell proliferative disordersaffecting breast cells. Cell proliferative disorders of the breast caninclude breast cancer, a precancer or precancerous condition of thebreast, benign growths or lesions of the breast, and malignant growthsor lesions of the breast, and metastatic lesions in tissue and organs inthe body other than the breast. Cell proliferative disorders of thebreast can include hyperplasia, metaplasia, and dysplasia of the breast.

A cell proliferative disorder of the breast can be a precancerouscondition of the breast. Compositions of the present invention may beused to treat a precancerous condition of the breast. A precancerouscondition of the breast can include atypical hyperplasia of the breast,ductal carcinoma in situ (DCIS), intraductal carcinoma, lobularcarcinoma in situ (LCIS), lobular neoplasia, and stage 0 or grade 0growth or lesion of the breast (e.g., stage 0 or grade 0 breast cancer,or carcinoma in situ). A precancerous condition of the breast can bestaged according to the TNM classification scheme as accepted by theAmerican Joint Committee on Cancer (AJCC), where the primary tumor (T)has been assigned a stage of T0 or Tis; and where the regional lymphnodes (N) have been assigned a stage of NO; and where distant metastasis(M) has been assigned a stage of M0.

The cell proliferative disorder of the breast can be breast cancer.Preferably, compositions of the present invention may be used to treatbreast cancer. Breast cancer includes all forms of cancer of the breast.Breast cancer can include primary epithelial breast cancers. Breastcancer can include cancers in which the breast is involved by othertumors such as lymphoma, sarcoma or melanoma. Breast cancer can includecarcinoma of the breast, ductal carcinoma of the breast, lobularcarcinoma of the breast, undifferentiated carcinoma of the breast,cystosarcoma phyllodes of the breast, angiosarcoma of the breast, andprimary lymphoma of the breast. Breast cancer can include Stage I, II,IIIA, IIIB, IIIC and IV breast cancer. Ductal carcinoma of the breastcan include invasive carcinoma, invasive carcinoma in situ withpredominant intraductal component, inflammatory breast cancer, and aductal carcinoma of the breast with a histologic type selected from thegroup consisting of comedo, mucinous (colloid), medullary, medullarywith lymphcytic infiltrate, papillary, scirrhous, and tubular. Lobularcarcinoma of the breast can include invasive lobular carcinoma withpredominant in situ component, invasive lobular carcinoma, andinfiltrating lobular carcinoma. Breast cancer can include Paget'sdisease, Paget's disease with intraductal carcinoma, and Paget's diseasewith invasive ductal carcinoma. Breast cancer can include breastneoplasms having histologic and ultrastructual heterogeneity (e.g.,mixed cell types).

Preferably, compound of the present invention, or a pharmaceuticallyacceptable salt, prodrug, metabolite, crystalline form, or solvatethereof, may be used to treat breast cancer. A breast cancer that is tobe treated can include familial breast cancer. A breast cancer that isto be treated can include sporadic breast cancer. A breast cancer thatis to be treated can arise in a male subject. A breast cancer that is tobe treated can arise in a female subject. A breast cancer that is to betreated can arise in a premenopausal female subject or a postmenopausalfemale subject. A breast cancer that is to be treated can arise in asubject equal to or older than 30 years old, or a subject younger than30 years old. A breast cancer that is to be treated has arisen in asubject equal to or older than 50 years old, or a subject younger than50 years old. A breast cancer that is to be treated can arise in asubject equal to or older than 70 years old, or a subject younger than70 years old.

A breast cancer that is to be treated can be typed to identify afamilial or spontaneous mutation in BRCA1, BRCA2, or p53. A breastcancer that is to be treated can be typed as having a HER2/neu geneamplification, as overexpressing HER2/neu, or as having a low,intermediate or high level of HER2/neu expression. A breast cancer thatis to be treated can be typed for a marker selected from the groupconsisting of estrogen receptor (ER), progesterone receptor (PR), humanepidermal growth factor receptor-2, Ki-67, CA15-3, CA 27-29, and c-Met.A breast cancer that is to be treated can be typed as ER-unknown,ER-rich or ER-poor. A breast cancer that is to be treated can be typedas ER-negative or ER-positive. ER-typing of a breast cancer may beperformed by any reproducible means. ER-typing of a breast cancer may beperformed as set forth in Onkologie 27: 175-179 (2004). A breast cancerthat is to be treated can be typed as PR-unknown, PR-rich, or PR-poor. Abreast cancer that is to be treated can be typed as PR-negative orPR-positive. A breast cancer that is to be treated can be typed asreceptor positive or receptor negative. A breast cancer that is to betreated can be typed as being associated with elevated blood levels ofCA 15-3, or CA 27-29, or both.

A breast cancer that is to be treated can include a localized tumor ofthe breast. A breast cancer that is to be treated can include a tumor ofthe breast that is associated with a negative sentinel lymph node (SLN)biopsy. A breast cancer that is to be treated can include a tumor of thebreast that is associated with a positive sentinel lymph node (SLN)biopsy. A breast cancer that is to be treated can include a tumor of thebreast that is associated with one or more positive axillary lymphnodes, where the axillary lymph nodes have been staged by any applicablemethod. A breast cancer that is to be treated can include a tumor of thebreast that has been typed as having nodal negative status (e.g.,node-negative) or nodal positive status (e.g., node-positive). A breastcancer that is to be treated can include a tumor of the breast that hasmetastasized to other locations in the body. A breast cancer that is tobe treated can be classified as having metastasized to a locationselected from the group consisting of bone, lung, liver, or brain. Abreast cancer that is to be treated can be classified according to acharacteristic selected from the group consisting of metastatic,localized, regional, local-regional, locally advanced, distant,multicentric, bilateral, ipsilateral, contralateral, newly diagnosed,recurrent, and inoperable.

A compound of the present invention, or a pharmaceutically acceptablesalt, prodrug, metabolite, crystalline form or solvate thereof, may beused to treat or prevent a cell proliferative disorder of the breast, orto treat or prevent breast cancer, in a subject having an increased riskof developing breast cancer relative to the population at large. Asubject with an increased risk of developing breast cancer relative tothe population at large is a female subject with a family history orpersonal history of breast cancer. A subject with an increased risk ofdeveloping breast cancer relative to the population at large is a femalesubject having a germ-line or spontaneous mutation in BRCA1 or BRCA2, orboth. A subject with an increased risk of developing breast cancerrelative to the population at large is a female subject with a familyhistory of breast cancer and a germ-line or spontaneous mutation inBRCA1 or BRCA2, or both. A subject with an increased risk of developingbreast cancer relative to the population at large is a female who isgreater than 30 years old, greater than 40 years old, greater than 50years old, greater than 60 years old, greater than 70 years old, greaterthan 80 years old, or greater than 90 years old. A subject with anincreased risk of developing breast cancer relative to the population atlarge is a subject with atypical hyperplasia of the breast, ductalcarcinoma in situ (DCIS), intraductal carcinoma, lobular carcinoma insitu (LCIS), lobular neoplasia, or a stage 0 growth or lesion of thebreast (e.g., stage 0 or grade 0 breast cancer, or carcinoma in situ).

A breast cancer that is to be treated can histologically gradedaccording to the Scarff-Bloom-Richardson system, wherein a breast tumorhas been assigned a mitosis count score of 1, 2, or 3; a nuclearpleiomorphism score of 1, 2, or 3; a tubule formation score of 1, 2, or3; and a total Scarff-Bloom-Richardson score of between 3 and 9. Abreast cancer that is to be treated can be assigned a tumor gradeaccording to the International Consensus Panel on the Treatment ofBreast Cancer selected from the group consisting of grade 1, grade 1-2,grade 2, grade 2-3, or grade 3.

A cancer that is to be treated can be staged according to the AmericanJoint Committee on Cancer (AJCC) TNM classification system, where thetumor (T) has been assigned a stage of TX, T1, T1mic, T1a, T1b, T1c, T₂,T₃, T₄, T₄a, T₄b, T₄c, or T₄d; and where the regional lymph nodes (N)have been assigned a stage of NX, N0, N1, N2, N2a, N2b, N3, N3a, N3b, orN3c; and where distant metastasis (M) can be assigned a stage of MX, M0,or M1. A cancer that is to be treated can be staged according to anAmerican Joint Committee on Cancer (AJCC) classification as Stage I,Stage IIA, Stage IIB, Stage IIIA, Stage IIIB, Stage IIIC, or Stage IV. Acancer that is to be treated can be assigned a grade according to anAJCC classification as Grade GX (e.g., grade cannot be assessed), Grade1, Grade 2, Grade 3 or Grade 4. A cancer that is to be treated can bestaged according to an AJCC pathologic classification (pN) of pNX, pN0,PN0 (I−), PN0 (I+), PN0 (mol−), PN0 (mol+), PN1, PN1(mi), PN1a, PN1b,PN1c, pN2, pN2a, pN2b, pN3, pN3a, pN3b, or pN3c.

A cancer that is to be treated can include a tumor that has beendetermined to be less than or equal to about 2 centimeters in diameter.A cancer that is to be treated can include a tumor that has beendetermined to be from about 2 to about 5 centimeters in diameter. Acancer that is to be treated can include a tumor that has beendetermined to be greater than or equal to about 3 centimeters indiameter. A cancer that is to be treated can include a tumor that hasbeen determined to be greater than 5 centimeters in diameter. A cancerthat is to be treated can be classified by microscopic appearance aswell differentiated, moderately differentiated, poorly differentiated,or undifferentiated. A cancer that is to be treated can be classified bymicroscopic appearance with respect to mitosis count (e.g., amount ofcell division) or nuclear pleiomorphism (e.g., change in cells). Acancer that is to be treated can be classified by microscopic appearanceas being associated with areas of necrosis (e.g., areas of dying ordegenerating cells). A cancer that is to be treated can be classified ashaving an abnormal karyotype, having an abnormal number of chromosomes,or having one or more chromosomes that are abnormal in appearance. Acancer that is to be treated can be classified as being aneuploid,triploid, tetraploid, or as having an altered ploidy. A cancer that isto be treated can be classified as having a chromosomal translocation,or a deletion or duplication of an entire chromosome, or a region ofdeletion, duplication or amplification of a portion of a chromosome.

A cancer that is to be treated can be evaluated by DNA cytometry, flowcytometry, or image cytometry. A cancer that is to be treated can betyped as having 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of cellsin the synthesis stage of cell division (e.g., in S phase of celldivision). A cancer that is to be treated can be typed as having a lowS-phase fraction or a high S-phase fraction.

As used herein, a “normal cell” is a cell that cannot be classified aspart of a “cell proliferative disorder”. A normal cell lacks unregulatedor abnormal growth, or both, that can lead to the development of anunwanted condition or disease. Preferably, a normal cell possessesnormally functioning cell cycle checkpoint control mechanisms.

As used herein, “contacting a cell” refers to a condition in which acompound or other composition of matter is in direct contact with acell, or is close enough to induce a desired biological effect in acell.

As used herein, “candidate compound” refers to a compound of the presentinvention, or a pharmaceutically acceptable salt, prodrug, metabolite,crystalline form or solvate thereof, that has been or will be tested inone or more in vitro or in vivo biological assays, in order to determineif that compound is likely to elicit a desired biological or medicalresponse in a cell, tissue, system, animal or human that is being soughtby a researcher or clinician. A candidate compound is a compound of thepresent invention, or a pharmaceutically acceptable salt, prodrug,metabolite, crystalline form or solvate thereof. The biological ormedical response can be the treatment of cancer. The biological ormedical response can be treatment or prevention of a cell proliferativedisorder. In vitro or in vivo biological assays can include, but are notlimited to, enzymatic activity assays, electrophoretic mobility shiftassays, reporter gene assays, in vitro cell viability assays, and theassays described herein.

As used herein, “monotherapy” refers to the administration of a singleactive or therapeutic compound to a subject in need thereof. Preferably,monotherapy will involve administration of a therapeutically effectiveamount of an single active compound. For example, cancer monotherapywith one of the compound of the present invention, or a pharmaceuticallyacceptable salt, prodrug, metabolite, analog or derivative thereof, to asubject in need of treatment of cancer. In one aspect, the single activecompound is a compound of the present invention, or a pharmaceuticallyacceptable salt, prodrug, metabolite, crystalline form or solvatethereof.

As used herein, “treating” or “treat” describes the management and careof a patient for the purpose of combating a disease, condition, ordisorder and includes the administration of a compound of the presentinvention, or a pharmaceutically acceptable salt, prodrug, metabolite,crystalline form or solvate thereof, to alleviate the symptoms orcomplications of a disease, condition or disorder, or to eliminate thedisease, condition or disorder.

A compound of the present invention, or a pharmaceutically acceptablesalt, prodrug, metabolite, crystalline form or solvate thereof, can alsobe used to prevent a disease, condition or disorder. As used herein,“preventing” or “prevent” describes reducing or eliminating the onset ofthe symptoms or complications of the disease, condition or disorder.

As used herein, the term “alleviate” is meant to describe a process bywhich the severity of a sign or symptom of a disorder is decreased.Importantly, a sign or symptom can be alleviated without beingeliminated. In a preferred embodiment, the administration ofpharmaceutical compositions of the invention leads to the elimination ofa sign or symptom, however, elimination is not required. Effectivedosages are expected to decrease the severity of a sign or symptom. Forinstance, a sign or symptom of a disorder such as cancer, which canoccur in multiple locations, is alleviated if the severity of the canceris decreased within at least one of multiple locations.

As used herein, the term “severity” is meant to describe the potentialof cancer to transform from a precancerous, or benign, state into amalignant state. Alternatively, or in addition, severity is meant todescribe a cancer stage, for example, according to the TNM system(accepted by the International Union Against Cancer (UICC) and theAmerican Joint Committee on Cancer (AJCC)) or by other art-recognizedmethods. Cancer stage refers to the extent or severity of the cancer,based on factors such as the location of the primary tumor, tumor size,number of tumors, and lymph node involvement (spread of cancer intolymph nodes). Alternatively, or in addition, severity is meant todescribe the tumor grade by art-recognized methods (see, National CancerInstitute, www.cancer.gov). Tumor grade is a system used to classifycancer cells in terms of how abnormal they look under a microscope andhow quickly the tumor is likely to grow and spread. Many factors areconsidered when determining tumor grade, including the structure andgrowth pattern of the cells. The specific factors used to determinetumor grade vary with each type of cancer. Severity also describes ahistologic grade, also called differentiation, which refers to how muchthe tumor cells resemble normal cells of the same tissue type (see,National Cancer Institute, www.cancer.gov). Furthermore, severitydescribes a nuclear grade, which refers to the size and shape of thenucleus in tumor cells and the percentage of tumor cells that aredividing (see, National Cancer Institute, www.cancer.gov).

In another aspect of the invention, severity describes the degree towhich a tumor has secreted growth factors, degraded the extracellularmatrix, become vascularized, lost adhesion to juxtaposed tissues, ormetastasized. Moreover, severity describes the number of locations towhich a primary tumor has metastasized. Finally, severity includes thedifficulty of treating tumors of varying types and locations. Forexample, inoperable tumors, those cancers which have greater access tomultiple body systems (hematological and immunological tumors), andthose which are the most resistant to traditional treatments areconsidered most severe. In these situations, prolonging the lifeexpectancy of the subject and/or reducing pain, decreasing theproportion of cancerous cells or restricting cells to one system, andimproving cancer stage/tumor grade/histological grade/nuclear grade areconsidered alleviating a sign or symptom of the cancer.

As used herein the term “symptom” is defined as an indication ofdisease, illness, injury, or that something is not right in the body.Symptoms are felt or noticed by the individual experiencing the symptom,but may not easily be noticed by others. Others are defined asnon-health-care professionals.

As used herein the term “sign” is also defined as an indication thatsomething is not right in the body. But signs are defined as things thatcan be seen by a doctor, nurse, or other health care professional.

Cancer is a group of diseases that may cause almost any sign or symptom.The signs and symptoms will depend on where the cancer is, the size ofthe cancer, and how much it affects the nearby organs or structures. Ifa cancer spreads (metastasizes), then symptoms may appear in differentparts of the body.

As a cancer grows, it begins to push on nearby organs, blood vessels,and nerves. This pressure creates some of the signs and symptoms ofcancer. If the cancer is in a critical area, such as certain parts ofthe brain, even the smallest tumor can cause early symptoms.

But sometimes cancers start in places where it does not cause anysymptoms until the cancer has grown quite large. Pancreas cancers, forexample, do not usually grow large enough to be felt from the outside ofthe body. Some pancreatic cancers do not cause symptoms until they beginto grow around nearby nerves (this causes a backache). Others growaround the bile duct, which blocks the flow of bile and leads to ayellowing of the skin known as jaundice. By the time a pancreatic cancercauses these signs or symptoms, it has usually reached an advancedstage.

A cancer may also cause symptoms such as fever, fatigue, or weight loss.This may be because cancer cells use up much of the body's energy supplyor release substances that change the body's metabolism. Or the cancermay cause the immune system to react in ways that produce thesesymptoms.

Sometimes, cancer cells release substances into the bloodstream thatcause symptoms not usually thought to result from cancers. For example,some cancers of the pancreas can release substances which cause bloodclots to develop in veins of the legs. Some lung cancers makehormone-like substances that affect blood calcium levels, affectingnerves and muscles and causing weakness and dizziness

Cancer presents several general signs or symptoms that occur when avariety of subtypes of cancer cells are present. Most people with cancerwill lose weight at some time with their disease. An unexplained(unintentional) weight loss of 10 pounds or more may be the first signof cancer, particularly cancers of the pancreas, stomach, esophagus, orlung.

Fever is very common with cancer, but is more often seen in advanceddisease. Almost all patients with cancer will have fever at some time,especially if the cancer or its treatment affects the immune system andmakes it harder for the body to fight infection. Less often, fever maybe an early sign of cancer, such as with leukemia or lymphoma.

Fatigue may be an important symptom as cancer progresses. It may happenearly, though, in cancers such as with leukemia, or if the cancer iscausing an ongoing loss of blood, as in some colon or stomach cancers.

Pain may be an early symptom with some cancers such as bone cancers ortesticular cancer. But most often pain is a symptom of advanced disease.

Along with cancers of the skin (see next section), some internal cancerscan cause skin signs that can be seen. These changes include the skinlooking darker (hyperpigmentation), yellow (jaundice), or red(erythema); itching; or excessive hair growth.

Alternatively, or in addition, cancer subtypes present specific signs orsymptoms. Changes in bowel habits or bladder function could indicatecancer. Long-term constipation, diarrhea, or a change in the size of thestool may be a sign of colon cancer. Pain with urination, blood in theurine, or a change in bladder function (such as more frequent or lessfrequent urination) could be related to bladder or prostate cancer.

Changes in skin condition or appearance of a new skin condition couldindicate cancer. Skin cancers may bleed and look like sores that do notheal. A long-lasting sore in the mouth could be an oral cancer,especially in patients who smoke, chew tobacco, or frequently drinkalcohol. Sores on the penis or vagina may either be signs of infectionor an early cancer.

Unusual bleeding or discharge could indicate cancer. Unusual bleedingcan happen in either early or advanced cancer. Blood in the sputum(phlegm) may be a sign of lung cancer. Blood in the stool (or a dark orblack stool) could be a sign of colon or rectal cancer. Cancer of thecervix or the endometrium (lining of the uterus) can cause vaginalbleeding. Blood in the urine may be a sign of bladder or kidney cancer.A bloody discharge from the nipple may be a sign of breast cancer.

A thickening or lump in the breast or in other parts of the body couldindicate the presence of a cancer. Many cancers can be felt through theskin, mostly in the breast, testicle, lymph nodes (glands), and the softtissues of the body. A lump or thickening may be an early or late signof cancer. Any lump or thickening could be indicative of cancer,especially if the formation is new or has grown in size.

Indigestion or trouble swallowing could indicate cancer. While thesesymptoms commonly have other causes, indigestion or swallowing problemsmay be a sign of cancer of the esophagus, stomach, or pharynx (throat).

Recent changes in a wart or mole could be indicative of cancer. Anywart, mole, or freckle that changes in color, size, or shape, or losesits definite borders indicates the potential development of cancer. Forexample, the skin lesion may be a melanoma.

A persistent cough or hoarseness could be indicative of cancer. A coughthat does not go away may be a sign of lung cancer. Hoarseness can be asign of cancer of the larynx (voice box) or thyroid.

While the signs and symptoms listed above are the more common ones seenwith cancer, there are many others that are less common and are notlisted here. However, all art-recognized signs and symptoms of cancerare contemplated and encompassed by the instant invention.

Treating cancer can result in a reduction in size of a tumor. Areduction in size of a tumor may also be referred to as “tumorregression”. Preferably, after treatment, tumor size is reduced by 5% orgreater relative to its size prior to treatment; more preferably, tumorsize is reduced by 10% or greater; more preferably, reduced by 20% orgreater; more preferably, reduced by 30% or greater; more preferably,reduced by 40% or greater; even more preferably, reduced by 50% orgreater; and most preferably, reduced by greater than 75% or greater.Size of a tumor may be measured by any reproducible means ofmeasurement. The size of a tumor may be measured as a diameter of thetumor.

Treating cancer can result in a reduction in tumor volume. Preferably,after treatment, tumor volume is reduced by 5% or greater relative toits size prior to treatment; more preferably, tumor volume is reduced by10% or greater; more preferably, reduced by 20% or greater; morepreferably, reduced by 30% or greater; more preferably, reduced by 40%or greater; even more preferably, reduced by 50% or greater; and mostpreferably, reduced by greater than 75% or greater. Tumor volume may bemeasured by any reproducible means of measurement.

Treating cancer results in a decrease in number of tumors. Preferably,after treatment, tumor number is reduced by 5% or greater relative tonumber prior to treatment; more preferably, tumor number is reduced by10% or greater; more preferably, reduced by 20% or greater; morepreferably, reduced by 30% or greater; more preferably, reduced by 40%or greater; even more preferably, reduced by 50% or greater; and mostpreferably, reduced by greater than 75%. Number of tumors may bemeasured by any reproducible means of measurement. The number of tumorsmay be measured by counting tumors visible to the naked eye or at aspecified magnification. Preferably, the specified magnification is 2×,3×, 4×, 5×, 10×, or 50×.

Treating cancer can result in a decrease in number of metastatic lesionsin other tissues or organs distant from the primary tumor site.Preferably, after treatment, the number of metastatic lesions is reducedby 5% or greater relative to number prior to treatment; more preferably,the number of metastatic lesions is reduced by 10% or greater; morepreferably, reduced by 20% or greater; more preferably, reduced by 30%or greater; more preferably, reduced by 40% or greater; even morepreferably, reduced by 50% or greater; and most preferably, reduced bygreater than 75%. The number of metastatic lesions may be measured byany reproducible means of measurement. The number of metastatic lesionsmay be measured by counting metastatic lesions visible to the naked eyeor at a specified magnification. Preferably, the specified magnificationis 2×, 3×, 4×, 5×, 10×, or 50×.

Treating cancer can result in an increase in average survival time of apopulation of treated subjects in comparison to a population receivingcarrier alone. Preferably, the average survival time is increased bymore than 30 days; more preferably, by more than 60 days; morepreferably, by more than 90 days; and most preferably, by more than 120days. An increase in average survival time of a population may bemeasured by any reproducible means. An increase in average survival timeof a population may be measured, for example, by calculating for apopulation the average length of survival following initiation oftreatment with an active compound. An increase in average survival timeof a population may also be measured, for example, by calculating for apopulation the average length of survival following completion of afirst round of treatment with an active compound.

Treating cancer can result in an increase in average survival time of apopulation of treated subjects in comparison to a population ofuntreated subjects. Preferably, the average survival time is increasedby more than 30 days; more preferably, by more than 60 days; morepreferably, by more than 90 days; and most preferably, by more than 120days. An increase in average survival time of a population may bemeasured by any reproducible means. An increase in average survival timeof a population may be measured, for example, by calculating for apopulation the average length of survival following initiation oftreatment with an active compound. An increase in average survival timeof a population may also be measured, for example, by calculating for apopulation the average length of survival following completion of afirst round of treatment with an active compound.

Treating cancer can result in increase in average survival time of apopulation of treated subjects in comparison to a population receivingmonotherapy with a drug that is not a compound of the present invention,or a pharmaceutically acceptable salt, prodrug, metabolite, analog orderivative thereof. Preferably, the average survival time is increasedby more than 30 days; more preferably, by more than 60 days; morepreferably, by more than 90 days; and most preferably, by more than 120days. An increase in average survival time of a population may bemeasured by any reproducible means. An increase in average survival timeof a population may be measured, for example, by calculating for apopulation the average length of survival following initiation oftreatment with an active compound. An increase in average survival timeof a population may also be measured, for example, by calculating for apopulation the average length of survival following completion of afirst round of treatment with an active compound.

Treating cancer can result in a decrease in the mortality rate of apopulation of treated subjects in comparison to a population receivingcarrier alone. Treating cancer can result in a decrease in the mortalityrate of a population of treated subjects in comparison to an untreatedpopulation. Treating cancer can result in a decrease in the mortalityrate of a population of treated subjects in comparison to a populationreceiving monotherapy with a drug that is not a compound of the presentinvention, or a pharmaceutically acceptable salt, prodrug, metabolite,analog or derivative thereof. Preferably, the mortality rate isdecreased by more than 2%; more preferably, by more than 5%; morepreferably, by more than 10%; and most preferably, by more than 25%. Adecrease in the mortality rate of a population of treated subjects maybe measured by any reproducible means. A decrease in the mortality rateof a population may be measured, for example, by calculating for apopulation the average number of disease-related deaths per unit timefollowing initiation of treatment with an active compound. A decrease inthe mortality rate of a population may also be measured, for example, bycalculating for a population the average number of disease-relateddeaths per unit time following completion of a first round of treatmentwith an active compound.

Treating cancer can result in a decrease in tumor growth rate.Preferably, after treatment, tumor growth rate is reduced by at least 5%relative to number prior to treatment; more preferably, tumor growthrate is reduced by at least 10%; more preferably, reduced by at least20%; more preferably, reduced by at least 30%; more preferably, reducedby at least 40%; more preferably, reduced by at least 50%; even morepreferably, reduced by at least 50%; and most preferably, reduced by atleast 75%. Tumor growth rate may be measured by any reproducible meansof measurement. Tumor growth rate can be measured according to a changein tumor diameter per unit time.

Treating cancer can result in a decrease in tumor regrowth. Preferably,after treatment, tumor regrowth is less than 5%; more preferably, tumorregrowth is less than 10%; more preferably, less than 20%; morepreferably, less than 30%; more preferably, less than 40%; morepreferably, less than 50%; even more preferably, less than 50%; and mostpreferably, less than 75%. Tumor regrowth may be measured by anyreproducible means of measurement. Tumor regrowth is measured, forexample, by measuring an increase in the diameter of a tumor after aprior tumor shrinkage that followed treatment. A decrease in tumorregrowth is indicated by failure of tumors to reoccur after treatmenthas stopped.

Treating or preventing a cell proliferative disorder can result in areduction in the rate of cellular proliferation. Preferably, aftertreatment, the rate of cellular proliferation is reduced by at least 5%;more preferably, by at least 10%; more preferably, by at least 20%; morepreferably, by at least 30%; more preferably, by at least 40%; morepreferably, by at least 50%; even more preferably, by at least 50%; andmost preferably, by at least 75%. The rate of cellular proliferation maybe measured by any reproducible means of measurement. The rate ofcellular proliferation is measured, for example, by measuring the numberof dividing cells in a tissue sample per unit time.

Treating or preventing a cell proliferative disorder can result in areduction in the proportion of proliferating cells. Preferably, aftertreatment, the proportion of proliferating cells is reduced by at least5%; more preferably, by at least 10%; more preferably, by at least 20%;more preferably, by at least 30%; more preferably, by at least 40%; morepreferably, by at least 50%; even more preferably, by at least 50%; andmost preferably, by at least 75%. The proportion of proliferating cellsmay be measured by any reproducible means of measurement. Preferably,the proportion of proliferating cells is measured, for example, byquantifying the number of dividing cells relative to the number ofnondividing cells in a tissue sample. The proportion of proliferatingcells can be equivalent to the mitotic index.

Treating or preventing a cell proliferative disorder can result in adecrease in size of an area or zone of cellular proliferation.Preferably, after treatment, size of an area or zone of cellularproliferation is reduced by at least 5% relative to its size prior totreatment; more preferably, reduced by at least 10%; more preferably,reduced by at least 20%; more preferably, reduced by at least 30%; morepreferably, reduced by at least 40%; more preferably, reduced by atleast 50%; even more preferably, reduced by at least 50%; and mostpreferably, reduced by at least 75%. Size of an area or zone of cellularproliferation may be measured by any reproducible means of measurement.The size of an area or zone of cellular proliferation may be measured asa diameter or width of an area or zone of cellular proliferation.

Treating or preventing a cell proliferative disorder can result in adecrease in the number or proportion of cells having an abnormalappearance or morphology. Preferably, after treatment, the number ofcells having an abnormal morphology is reduced by at least 5% relativeto its size prior to treatment; more preferably, reduced by at least10%; more preferably, reduced by at least 20%; more preferably, reducedby at least 30%; more preferably, reduced by at least 40%; morepreferably, reduced by at least 50%; even more preferably, reduced by atleast 50%; and most preferably, reduced by at least 75%. An abnormalcellular appearance or morphology may be measured by any reproduciblemeans of measurement. An abnormal cellular morphology can be measured bymicroscopy, e.g., using an inverted tissue culture microscope. Anabnormal cellular morphology can take the form of nuclear pleiomorphism.

As used herein, the term “selectively” means tending to occur at ahigher frequency in one population than in another population. Thecompared populations can be cell populations. Preferably, a compound ofthe present invention, or a pharmaceutically acceptable salt, prodrug,metabolite, crystalline form or solvate thereof, acts selectively on acancer or precancerous cell but not on a normal cell. Preferably, acompound of the present invention, or a pharmaceutically acceptablesalt, prodrug, metabolite, crystalline form or solvate thereof, actsselectively to modulate one molecular target (e.g., a target proteinmethyltransferase) but does not significantly modulate another moleculartarget (e.g., a non-target protein methyltransferase). The inventionalso provides a method for selectively inhibiting the activity of anenzyme, such as a protein methyltransferase. Preferably, an event occursselectively in population A relative to population B if it occursgreater than two times more frequently in population A as compared topopulation B. An event occurs selectively if it occurs greater than fivetimes more frequently in population A. An event occurs selectively if itoccurs greater than ten times more frequently in population A; morepreferably, greater than fifty times; even more preferably, greater than100 times; and most preferably, greater than 1000 times more frequentlyin population A as compared to population B. For example, cell deathwould be said to occur selectively in cancer cells if it occurredgreater than twice as frequently in cancer cells as compared to normalcells.

A compound of the present invention, or a pharmaceutically acceptablesalt, prodrug, metabolite, crystalline form or solvate thereof, canmodulate the activity of a molecular target (e.g., a target proteinmethyltransferase). Modulating refers to stimulating or inhibiting anactivity of a molecular target. Preferably, a compound of the presentinvention, or a pharmaceutically acceptable salt, prodrug, metabolite,crystalline form or solvate thereof, modulates the activity of amolecular target if it stimulates or inhibits the activity of themolecular target by at least 2-fold relative to the activity of themolecular target under the same conditions but lacking only the presenceof said compound. More preferably, a compound of the present invention,or a pharmaceutically acceptable salt, prodrug, metabolite, crystallineform or solvate thereof, modulates the activity of a molecular target ifit stimulates or inhibits the activity of the molecular target by atleast 5-fold, at least 10-fold, at least 20-fold, at least 50-fold, atleast 100-fold relative to the activity of the molecular target underthe same conditions but lacking only the presence of said compound. Theactivity of a molecular target may be measured by any reproduciblemeans. The activity of a molecular target may be measured in vitro or invivo. For example, the activity of a molecular target may be measured invitro by an enzymatic activity assay or a DNA binding assay, or theactivity of a molecular target may be measured in vivo by assaying forexpression of a reporter gene.

A compound of the present invention, or a pharmaceutically acceptablesalt, prodrug, metabolite, crystalline form or solvate thereof, does notsignificantly modulate the activity of a molecular target if theaddition of the compound does not stimulate or inhibit the activity ofthe molecular target by greater than 10% relative to the activity of themolecular target under the same conditions but lacking only the presenceof said compound.

As used herein, the term “isozyme selective” means preferentialinhibition or stimulation of a first isoform of an enzyme in comparisonto a second isoform of an enzyme (e.g., preferential inhibition orstimulation of a protein methyltransferase isozyme alpha in comparisonto a protein methyltransferase isozyme beta). Preferably, a compound ofthe present invention, or a pharmaceutically acceptable salt, prodrug,metabolite, crystalline form or solvate thereof, demonstrates a minimumof a fourfold differential, preferably a tenfold differential, morepreferably a fifty fold differential, in the dosage required to achievea biological effect. Preferably, a compound of the present invention, ora pharmaceutically acceptable salt, prodrug, metabolite, crystallineform or solvate thereof, demonstrates this differential across the rangeof inhibition, and the differential is exemplified at the IC₅₀, i.e., a50% inhibition, for a molecular target of interest.

Administering a compound of the present invention, or a pharmaceuticallyacceptable salt, prodrug, metabolite, crystalline form or solvatethereof, to a cell or a subject in need thereof can result in modulation(i.e., stimulation or inhibition) of an activity of a proteinmethyltransferase of interest.

The present invention provides methods to assess biological activity ofa compound of the present invention, or a pharmaceutically acceptablesalt, prodrug, metabolite, crystalline form or solvate thereof ormethods of identifying a test compound as a modulator (e.g., aninhibitor) of DOT1L. DOT1L polypeptides and nucleic acids can be used toscreen for compounds that bind to and/or modulate (e.g., increase ordecrease) one or more biological activities of DOT1L, including but notlimited to H3K79 HMTase activity, SAM binding activity, histone and/ornucleosome binding activity, AF10 binding activity, AF10-MLL or otherMLL fusion protein binding activity, and/or any other biologicalactivity of interest. A DOT1L polypeptide can be a functional fragmentof a full-length DOT1L polypeptide or functional equivalent thereof, andmay comprise any DOT1 domain of interest, including but not limited tothe catalytic domain, the SAM binding domain and/or the positivelycharged domain, the AF10 interaction domain and/or a nuclear exportsignal.

Methods of assessing DOT1L binding to histones, nucleosomes, nucleicacids or polypeptides can be carried out using standard techniques thatwill be apparent to those skilled in the art (see the Exemplificationfor exemplary methods). Such methods include yeast and mammaliantwo-hybrid assays and co-immunoprecipitation techniques.

For example, a compound that modulates DOT1L H3K79 HMTase activity canbe verified by: contacting a DOT1L polypeptide with a histone or peptidesubstrate comprising H3 in the presence of a test compound; detectingthe level of H3K79 methylation of the histone or peptide substrate underconditions sufficient to provide H3K79 methylation, wherein an elevationor reduction in H3K79 methylation in the presence of the test compoundas compared with the level of histone H3K79 methylation in the absenceof the test compound indicates that the test compound modulates DOT1LH3K79 HMTase activity.

The screening methods of the invention can be carried out in acell-based or cell-free system. As a further alternative, the assay canbe performed in a whole animal (including transgenic non-human animals).Further, with respect to cell-based systems, the DOT1L polypeptide (orany other polypeptide used in the assay) can be added directly to thecell or can be produced from a nucleic acid in the cell. The nucleicacid can be endogenous to the cell or can be foreign (e.g., agenetically modified cell).

In some assays, immunological reagents, e.g., antibodies and antigens,are employed. Fluorescence can be utilized in the measurement ofenzymatic activity in some assays. As used herein, “fluorescence” refersto a process through which a molecule emits a photon as a result ofabsorbing an incoming photon of higher energy by the same molecule.Specific methods for assessing the biological activity of the disclosedcompounds are described in the examples.

Administering a compound of the present invention, or a pharmaceuticallyacceptable salt, prodrug, metabolite, crystalline form or solvatethereof, to a cell or a subject in need thereof results in modulation(i.e., stimulation or inhibition) of an activity of an intracellulartarget (e.g., substrate). Several intracellular targets can be modulatedwith the compounds of the present invention, including, but not limitedto, protein methyltrasferase.

Activating refers to placing a composition of matter (e.g., protein ornucleic acid) in a state suitable for carrying out a desired biologicalfunction. A composition of matter capable of being activated also has anunactivated state. An activated composition of matter may have aninhibitory or stimulatory biological function, or both.

Elevation refers to an increase in a desired biological activity of acomposition of matter (e.g., a protein or a nucleic acid). Elevation mayoccur through an increase in concentration of a composition of matter.

As used herein, “a cell cycle checkpoint pathway” refers to abiochemical pathway that is involved in modulation of a cell cyclecheckpoint. A cell cycle checkpoint pathway may have stimulatory orinhibitory effects, or both, on one or more functions comprising a cellcycle checkpoint. A cell cycle checkpoint pathway is comprised of atleast two compositions of matter, preferably proteins, both of whichcontribute to modulation of a cell cycle checkpoint. A cell cyclecheckpoint pathway may be activated through an activation of one or moremembers of the cell cycle checkpoint pathway. Preferably, a cell cyclecheckpoint pathway is a biochemical signaling pathway.

As used herein, “cell cycle checkpoint regulator” refers to acomposition of matter that can function, at least in part, in modulationof a cell cycle checkpoint. A cell cycle checkpoint regulator may havestimulatory or inhibitory effects, or both, on one or more functionscomprising a cell cycle checkpoint. A cell cycle checkpoint regulatorcan be a protein or not a protein.

Treating cancer or a cell proliferative disorder can result in celldeath, and preferably, cell death results in a decrease of at least 10%in number of cells in a population. More preferably, cell death means adecrease of at least 20%; more preferably, a decrease of at least 30%;more preferably, a decrease of at least 40%; more preferably, a decreaseof at least 50%; most preferably, a decrease of at least 75%. Number ofcells in a population may be measured by any reproducible means. Anumber of cells in a population can be measured by fluorescenceactivated cell sorting (FACS), immunofluorescence microscopy and lightmicroscopy. Methods of measuring cell death are as shown in Li et al.,Proc Natl Acad Sci USA. 100(5): 2674-8, 2003. In an aspect, cell deathoccurs by apoptosis.

Preferably, an effective amount of a compound of the present invention,or a pharmaceutically acceptable salt, prodrug, metabolite, crystallineform or solvate thereof, is not significantly cytotoxic to normal cells.A therapeutically effective amount of a compound is not significantlycytotoxic to normal cells if administration of the compound in atherapeutically effective amount does not induce cell death in greaterthan 10% of normal cells. A therapeutically effective amount of acompound does not significantly affect the viability of normal cells ifadministration of the compound in a therapeutically effective amountdoes not induce cell death in greater than 10% of normal cells. In anaspect, cell death occurs by apoptosis.

Contacting a cell with a compound of the present invention, or apharmaceutically acceptable salt, prodrug, metabolite, crystalline formor solvate thereof, can induce or activate cell death selectively incancer cells. Administering to a subject in need thereof a compound ofthe present invention, or a pharmaceutically acceptable salt, prodrug,metabolite, crystalline form or solvate thereof, can induce or activatecell death selectively in cancer cells. Contacting a cell with acompound of the present invention, or a pharmaceutically acceptablesalt, prodrug, metabolite, crystalline form or solvate thereof, caninduce cell death selectively in one or more cells affected by a cellproliferative disorder. Preferably, administering to a subject in needthereof a compound of the present invention, or a pharmaceuticallyacceptable salt, prodrug, metabolite, crystalline form or solvatethereof, induces cell death selectively in one or more cells affected bya cell proliferative disorder.

The present invention relates to a method of treating or preventingcancer by administering a compound of the present invention, or apharmaceutically acceptable salt, prodrug, metabolite, crystalline formor solvate thereof, to a subject in need thereof, where administrationof the compound of the present invention, or a pharmaceuticallyacceptable salt, prodrug, metabolite, crystalline form or solvatethereof, results in one or more of the following: accumulation of cellsin G1 and/or S phase of the cell cycle, cytotoxicity via cell death incancer cells without a significant amount of cell death in normal cells,antitumor activity in animals with a therapeutic index of at least 2,and activation of a cell cycle checkpoint. As used herein, “therapeuticindex” is the maximum tolerated dose divided by the efficacious dose.

One skilled in the art may refer to general reference texts for detaileddescriptions of known techniques discussed herein or equivalenttechniques. These texts include Ausubel et al., Current Protocols inMolecular Biology, John Wiley and Sons, Inc. (2005); Sambrook et al.,Molecular Cloning. A Laboratory Manual (3^(rd) edition), Cold SpringHarbor Press, Cold Spring Harbor, N.Y. (2000); Coligan et al., CurrentProtocols in Immunology, John Wiley & Sons, N.Y.; Enna et al., CurrentProtocols in Pharmacology, John Wiley & Sons, N.Y.; Fingl et al., ThePharmacological Basis of Therapeutics (1975), Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa., 18^(th) edition (1990).These texts can, of course, also be referred to in making or using anaspect of the invention

The compounds of the instant invention can also be utilized to treat orprevent neurologic diseases or disorders. Neurologic diseases ordisorders that may be treated with the compounds of this inventioninclude epilepsy, schizophrenia, bipolar disorder or other psychologicaland/or psychiatric disorders, neuropathies, skeletal muscle atrophy, andneurodegenerative diseases, e.g., a neurodegenerative disease. Exemplaryneurodegenerative diseases include: Alzheimer's, Amyotrophic LateralSclerosis (ALS), and Parkinson's disease. Another class ofneurodegenerative diseases includes diseases caused at least in part byaggregation of poly-glutamine. Diseases of this class include:Huntington's Diseases, Spinalbulbar Muscular Atrophy (SBMA or Kennedy'sDisease) Dentatorubropallidoluysian Atrophy (DRPLA), SpinocerebellarAtaxia 1 (SCA1), Spinocerebellar Ataxia 2 (SCA2), Machado-Joseph Disease(MJD; SCA3), Spinocerebellar Ataxia 6 (SCA6), Spinocerebellar Ataxia 7(SCA7), and Spinocerebellar Ataxia 12 (SCA12).

Any other disease in which epigenetic methylation, which is mediated byDOT1, plays a role may be treatable or preventable using compounds andmethods described herein.

The present invention also provides pharmaceutical compositionscomprising a compound of the invention in combination with at least onepharmaceutically acceptable excipient or carrier.

A “pharmaceutical composition” is a formulation containing the compoundsof the present invention in a form suitable for administration to asubject. In one embodiment, the pharmaceutical composition is in bulk orin unit dosage form. The unit dosage form is any of a variety of forms,including, for example, a capsule, an IV bag, a tablet, a single pump onan aerosol inhaler or a vial. The quantity of active ingredient (e.g., aformulation of the disclosed compound or salt, hydrate, solvate orisomer thereof) in a unit dose of composition is an effective amount andis varied according to the particular treatment involved. One skilled inthe art will appreciate that it is sometimes necessary to make routinevariations to the dosage depending on the age and condition of thepatient. The dosage will also depend on the route of administration. Avariety of routes are contemplated, including oral, pulmonary, rectal,parenteral, transdermal, subcutaneous, intravenous, intramuscular,intraperitoneal, inhalational, buccal, sublingual, intrapleural,intrathecal, intranasal, and the like. Dosage forms for the topical ortransdermal administration of a compound of this invention includepowders, sprays, ointments, pastes, creams, lotions, gels, solutions,patches and inhalants. In one embodiment, the active compound is mixedunder sterile conditions with a pharmaceutically acceptable carrier, andwith any preservatives, buffers, or propellants that are required.

As used herein, the phrase “pharmaceutically acceptable” refers to thosecompounds, materials, compositions, carriers, and/or dosage forms whichare, within the scope of sound medical judgment, suitable for use incontact with the tissues of human beings and animals without excessivetoxicity, irritation, allergic response, or other problem orcomplication, commensurate with a reasonable benefit/risk ratio.

“Pharmaceutically acceptable excipient” means an excipient that isuseful in preparing a pharmaceutical composition that is generally safe,non-toxic and neither biologically nor otherwise undesirable, andincludes excipient that is acceptable for veterinary use as well ashuman pharmaceutical use. A “pharmaceutically acceptable excipient” asused in the specification and claims includes both one and more than onesuch excipient.

A pharmaceutical composition of the invention is formulated to becompatible with its intended route of administration. Examples of routesof administration include parenteral, e.g., intravenous, intradermal,subcutaneous, oral (e.g., inhalation), transdermal (topical), andtransmucosal administration. Solutions or suspensions used forparenteral, intradermal, or subcutaneous application can include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates, and agents for theadjustment of tonicity such as sodium chloride or dextrose. The pH canbe adjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes or multiple dose vials made of glass or plastic.

A compound or pharmaceutical composition of the invention can beadministered to a subject in many of the well-known methods currentlyused for chemotherapeutic treatment. For example, for treatment ofcancers, a compound of the invention may be injected directly intotumors, injected into the blood stream or body cavities or taken orallyor applied through the skin with patches. The dose chosen should besufficient to constitute effective treatment but not as high as to causeunacceptable side effects. The state of the disease condition (e.g.,cancer, precancer, and the like) and the health of the patient shouldpreferably be closely monitored during and for a reasonable period aftertreatment.

The term “therapeutically effective amount”, as used herein, refers toan amount of a pharmaceutical agent to treat, ameliorate, or prevent anidentified disease or condition, or to exhibit a detectable therapeuticor inhibitory effect. The effect can be detected by any assay methodknown in the art. The precise effective amount for a subject will dependupon the subject's body weight, size, and health; the nature and extentof the condition; and the therapeutic selected for administration.Therapeutically effective amounts for a given situation can bedetermined by routine experimentation that is within the skill andjudgment of the clinician. In a preferred aspect, the disease orcondition to be treated is cancer. In another aspect, the disease orcondition to be treated is a cell proliferative disorder.

For any compound, the therapeutically effective amount can be estimatedinitially either in cell culture assays, e.g., of neoplastic cells, orin animal models, usually rats, mice, rabbits, dogs, or pigs. The animalmodel may also be used to determine the appropriate concentration rangeand route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.Therapeutic/prophylactic efficacy and toxicity may be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., ED₅₀ (the dose therapeutically effective in 50% of thepopulation) and LD₅₀ (the dose lethal to 50% of the population). Thedose ratio between toxic and therapeutic effects is the therapeuticindex, and it can be expressed as the ratio, LD₅₀/ED₅₀. Pharmaceuticalcompositions that exhibit large therapeutic indices are preferred. Thedosage may vary within this range depending upon the dosage formemployed, sensitivity of the patient, and the route of administration.

Dosage and administration are adjusted to provide sufficient levels ofthe active agent(s) or to maintain the desired effect. Factors which maybe taken into account include the severity of the disease state, generalhealth of the subject, age, weight, and gender of the subject, diet,time and frequency of administration, drug interaction(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on half-life and clearance rateof the particular formulation.

The pharmaceutical compositions containing active compounds of thepresent invention may be manufactured in a manner that is generallyknown, e.g., by means of conventional mixing, dissolving, granulating,dragee-making, levigating, emulsifying, encapsulating, entrapping, orlyophilizing processes. Pharmaceutical compositions may be formulated ina conventional manner using one or more pharmaceutically acceptablecarriers comprising excipients and/or auxiliaries that facilitateprocessing of the active compounds into preparations that can be usedpharmaceutically. Of course, the appropriate formulation is dependentupon the route of administration chosen.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. For intravenous administration, suitablecarriers include physiological saline, bacteriostatic water, CremophorEL™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In allcases, the composition must be sterile and should be fluid to the extentthat easy syringeability exists. It must be stable under the conditionsof manufacture and storage and must be preserved against thecontaminating action of microorganisms such as bacteria and fungi. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. The proper fluidity can be maintained, for example, by the useof a coating such as lecithin, by the maintenance of the requiredparticle size in the case of dispersion and by the use of surfactants.Prevention of the action of microorganisms can be achieved by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In manycases, it will be preferable to include isotonic agents, for example,sugars, polyalcohols such as manitol and sorbitol, and sodium chloridein the composition. Prolonged absorption of the injectable compositionscan be brought about by including in the composition an agent whichdelays absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions can be prepared by incorporating the activecompound in the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, methods of preparation are vacuum dryingand freeze-drying that yields a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

Oral compositions generally include an inert diluent or an ediblepharmaceutically acceptable carrier. They can be enclosed in gelatincapsules or compressed into tablets. For the purpose of oral therapeuticadministration, the active compound can be incorporated with excipientsand used in the form of tablets, troches, or capsules. Oral compositionscan also be prepared using a fluid carrier for use as a mouthwash,wherein the compound in the fluid carrier is applied orally and swishedand expectorated or swallowed. Pharmaceutically compatible bindingagents, and/or adjuvant materials can be included as part of thecomposition. The tablets, pills, capsules, troches and the like cancontain any of the following ingredients, or compounds of a similarnature: a binder such as microcrystalline cellulose, gum tragacanth orgelatin; an excipient such as starch or lactose, a disintegrating agentsuch as alginic acid, Primogel, or corn starch; a lubricant such asmagnesium stearate or Sterotes; a glidant such as colloidal silicondioxide; a sweetening agent such as sucrose or saccharin; or a flavoringagent such as peppermint, methyl salicylate, or orange flavoring.

For administration by inhalation, the compounds are delivered in theform of an aerosol spray from pressured container or dispenser, whichcontains a suitable propellant, e.g., a gas such as carbon dioxide, or anebulizer.

Systemic administration can also be by transmucosal or transdermalmeans. For transmucosal or transdermal administration, penetrantsappropriate to the barrier to be permeated are used in the formulation.Such penetrants are generally known in the art, and include, forexample, for transmucosal administration, detergents, bile salts, andfusidic acid derivatives. Transmucosal administration can beaccomplished through the use of nasal sprays or suppositories. Fortransdermal administration, the active compounds are formulated intoointments, salves, gels, or creams as generally known in the art.

The active compounds can be prepared with pharmaceutically acceptablecarriers that will protect the compound against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions(including liposomes targeted to infected cells with monoclonalantibodies to viral antigens) can also be used as pharmaceuticallyacceptable carriers. These can be prepared according to methods known tothose skilled in the art, for example, as described in U.S. Pat. No.4,522,811.

It is especially advantageous to formulate oral or parenteralcompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the subject tobe treated; each unit containing a predetermined quantity of activecompound calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier. The specificationfor the dosage unit forms of the invention are dictated by and directlydependent on the unique characteristics of the active compound and theparticular therapeutic effect to be achieved.

In therapeutic applications, the dosages of the pharmaceuticalcompositions used in accordance with the invention vary depending on theagent, the age, weight, and clinical condition of the recipient patient,and the experience and judgment of the clinician or practitioneradministering the therapy, among other factors affecting the selecteddosage. Generally, the dose should be sufficient to result in slowing,and preferably regressing, the growth of the tumors and also preferablycausing complete regression of the cancer. Dosages can range from about0.01 mg/kg per day to about 5000 mg/kg per day. In preferred aspects,dosages can range from about 1 mg/kg per day to about 1000 mg/kg perday. In an aspect, the dose will be in the range of about 0.1 mg/day toabout 50 g/day; about 0.1 mg/day to about 25 g/day; about 0.1 mg/day toabout 10 g/day; about 0.1 mg to about 3 g/day; or about 0.1 mg to about1 g/day, in single, divided, or continuous doses (which dose may beadjusted for the patient's weight in kg, body surface area in m², andage in years). An effective amount of a pharmaceutical agent is thatwhich provides an objectively identifiable improvement as noted by theclinician or other qualified observer. For example, regression of atumor in a patient may be measured with reference to the diameter of atumor. Decrease in the diameter of a tumor indicates regression.Regression is also indicated by failure of tumors to reoccur aftertreatment has stopped. As used herein, the term “dosage effectivemanner” refers to amount of an active compound to produce the desiredbiological effect in a subject or cell.

The pharmaceutical compositions can be included in a container, pack, ordispenser together with instructions for administration.

The compounds of the present invention are capable of further formingsalts. All of these forms are also contemplated within the scope of theclaimed invention.

As used herein, “pharmaceutically acceptable salts” refer to derivativesof the compounds of the present invention wherein the parent compound ismodified by making acid or base salts thereof. Examples ofpharmaceutically acceptable salts include, but are not limited to,mineral or organic acid salts of basic residues such as amines, alkalior organic salts of acidic residues such as carboxylic acids, and thelike. The pharmaceutically acceptable salts include the conventionalnon-toxic salts or the quaternary ammonium salts of the parent compoundformed, for example, from non-toxic inorganic or organic acids. Forexample, such conventional non-toxic salts include, but are not limitedto, those derived from inorganic and organic acids selected from2-acetoxybenzoic, 2-hydroxyethane sulfonic, acetic, ascorbic, benzenesulfonic, benzoic, bicarbonic, carbonic, citric, edetic, ethanedisulfonic, 1,2-ethane sulfonic, fumaric, glucoheptonic, gluconic,glutamic, glycolic, glycollyarsanilic, hexylresorcinic, hydrabamic,hydrobromic, hydrochloric, hydroiodic, hydroxymaleic, hydroxynaphthoic,isethionic, lactic, lactobionic, lauryl sulfonic, maleic, malic,mandelic, methane sulfonic, napsylic, nitric, oxalic, pamoic,pantothenic, phenylacetic, phosphoric, polygalacturonic, propionic,salicyclic, stearic, subacetic, succinic, sulfamic, sulfanilic,sulfuric, tannic, tartaric, toluene sulfonic, and the commonly occurringamine acids, e.g., glycine, alanine, phenylalanine, arginine, etc.

Other examples of pharmaceutically acceptable salts include hexanoicacid, cyclopentane propionic acid, pyruvic acid, malonic acid,3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, 4-chlorobenzenesulfonicacid, 2-naphthalenesulfonic acid, 4-toluenesulfonic acid,camphorsulfonic acid, 4-methylbicyclo-[2.2.2]-oct-2-ene-1-carboxylicacid, 3-phenylpropionic acid, trimethylacetic acid, tertiary butylaceticacid, muconic acid, and the like. The present invention also encompassessalts formed when an acidic proton present in the parent compound eitheris replaced by a metal ion, e.g., an alkali metal ion, an alkaline earthion, or an aluminum ion; or coordinates with an organic base such asethanolamine, diethanolamine, triethanolamine, tromethamine,N-methylglucamine, and the like.

It should be understood that all references to pharmaceuticallyacceptable salts include solvent addition forms (solvates) orcrystalline forms as defined herein, of the same salt.

The compounds of the present invention can also be prepared as esters,for example, pharmaceutically acceptable esters. For example, acarboxylic acid function group in a compound can be converted to itscorresponding ester, e.g., a methyl, ethyl or other ester. Also, analcohol group in a compound can be converted to its corresponding ester,e.g., acetate, propionate or other ester.

The compounds of the present invention can also be prepared as prodrugs,for example, pharmaceutically acceptable prodrugs. The terms “pro-drug”and “prodrug” are used interchangeably herein and refer to any compoundwhich releases an active parent drug in vivo. Since prodrugs are knownto enhance numerous desirable qualities of pharmaceuticals (e.g.,solubility, bioavailability, manufacturing, etc.), the compounds of thepresent invention can be delivered in prodrug form. Thus, the presentinvention is intended to cover prodrugs of the presently claimedcompounds, methods of delivering the same and compositions containingthe same. “Prodrugs” are intended to include any covalently bondedcarriers that release an active parent drug of the present invention invivo when such prodrug is administered to a subject. Prodrugs in thepresent invention are prepared by modifying functional groups present inthe compound in such a way that the modifications are cleaved, either inroutine manipulation or in vivo, to the parent compound. Prodrugsinclude compounds of the present invention wherein a hydroxy, amino,sulfhydryl, carboxy or carbonyl group is bonded to any group that may becleaved in vivo to form a free hydroxyl, free amino, free sulfhydryl,free carboxy or free carbonyl group, respectively.

Examples of prodrugs include, but are not limited to, esters (e.g.,acetate, dialkylaminoacetates, formates, phosphates, sulfates andbenzoate derivatives) and carbamates (e.g., N,N-dimethylaminocarbonyl)of hydroxy functional groups, esters (e.g., ethyl esters,morpholinoethanol esters) of carboxyl functional groups, N-acylderivatives (e.g., N-acetyl) N-Mannich bases, Schiff bases andenaminones of amino functional groups, oximes, acetals, ketals and enolesters of ketone and aldehyde functional groups in compounds of theinvention, and the like, See Bundegaard, H., Design of Prodrugs, p 1-92,Elesevier, New York-Oxford (1985).

The compounds, or pharmaceutically acceptable salts, esters or prodrugsthereof, are administered orally, nasally, transdermally, pulmonary,inhalationally, buccally, sublingually, intraperintoneally,subcutaneously, intramuscularly, intravenously, rectally,intrapleurally, intrathecally and parenterally. In one embodiment, thecompound is administered orally. One skilled in the art will recognizethe advantages of certain routes of administration.

The dosage regimen utilizing the compounds is selected in accordancewith a variety of factors including type, species, age, weight, sex andmedical condition of the patient; the severity of the condition to betreated; the route of administration; the renal and hepatic function ofthe patient; and the particular compound or salt thereof employed. Anordinarily skilled physician or veterinarian can readily determine andprescribe the effective amount of the drug required to prevent, counter,or arrest the progress of the condition.

Techniques for formulation and administration of the disclosed compoundsof the invention can be found in Remington: the Science and Practice ofPharmacy, 19^(th) edition, Mack Publishing Co., Easton, Pa. (1995). Inan embodiment, the compounds described herein, and the pharmaceuticallyacceptable salts thereof, are used in pharmaceutical preparations incombination with a pharmaceutically acceptable carrier or diluent.Suitable pharmaceutically acceptable carriers include inert solidfillers or diluents and sterile aqueous or organic solutions. Thecompounds will be present in such pharmaceutical compositions in amountssufficient to provide the desired dosage amount in the range describedherein.

All percentages and ratios used herein, unless otherwise indicated, areby weight. Other features and advantages of the present invention areapparent from the different examples. The provided examples illustratedifferent components and methodology useful in practicing the presentinvention. The examples do not limit the claimed invention. Based on thepresent disclosure the skilled artisan can identify and employ othercomponents and methodology useful for practicing the present invention.

In the synthetic schemes described herein, compounds may be drawn withone particular configuration for simplicity. Such particularconfigurations are not to be construed as limiting the invention to oneor another isomer, tautomer, regioisomer or stereoisomer, nor does itexclude mixtures of isomers, tautomers, regioisomers or stereoisomers.

Compounds described herein are assayed for modulation of activity, forexample, histone methylation, modulation of cell growth and/or IC₅₀,described in the examples below. IC₅₀ values are presented as A=<0.1 μM;B=>0.1 μM and <1 μM; C=>1 μM and <10 μM; and D=>10 μM and <50 μM.

DOT1L Compound IC₅₀ (μM) EP-1 (EPZ-5676) 0.00074 EPZ-5677 0.00073

Example 1: Synthesis of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol(EPZ-5676 or EP-1) hydrate

(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol(EPZ-5676 or EP-1) hydrate was synthesized according to the schemebelow.

Step 1: Synthesis of9-((3aR,4R,6R,6aR)-6-((isopropylamino)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-purin-6-amine(2)

The materials employed for Step 1 are as follows:

MW Grade and Reagents (density) Amount Mol. Equiv. Comment Compound 1306.32 1100 g 3.591 1.0 N/A Acetone 58.08 2.64 L 2.4 Vol ACS, ≥99.5%(0.791) Acetic acid 60.05 206 mL 3.591 1.0 ACS (1.049) Sodium 211.941525 g 7.182 1.9 95% triacetoxy borohydride (STAB) Solvents Methanol 8.8L   8 Vol ACS, ≥99.9% Workup Acetonitrile 40 L Chromasolv, ≥99.9%

To a 30 L 3-neck jacketed vessel with a mechanical stirrer, athermocouple, and a N₂ inlet were charged 1 (1100 g, 3.591 mol), acetone(2.64 L), acetic acid (206 mL), and methanol (8.8 L) at roomtemperature. The resulting mixture was stirred at room temperature for5-10 min until all solids were dissolved. The solution was cooled toabout 16-18° C., and STAB (305 g, 0.38 eq) was added over 1-2 min. Theaddition of STAB was moderately exothermic, the batch temperature shouldbe cooled to about 16-18° C. prior to the STAB addition, so that thereaction mixture temperature was kept below 25° C. The rest of STAB wasadded in 4 equal portions (305 g, 0.38 eq each) over next 2 h,maintaining the batch temperature between 20-25° C. The batch as asolution was stirred at the same temperature for an additional 1-2 h.The batch appeared as a light yellow solution with a little bit ofhaziness. At this stage the reaction should give a full conversionmonitored by HPLC. The reaction mixture was concentrated on rotavapunder vacuum to remove all acetone and methanol, flushed withacetonitrile (4.4 L×2). Some inorganic solids were precipitated outduring the concentration. The solid was removed by filtration, the wetcake was washed with MeCN (4.4 L). The combined filtrate wasconcentrated, flushed with MeCN (4.4 L). There was no 2 trapped in theinorganic solid. MeCN (18-20 L) was added to the concentrated oil, theresulting solution was analyzed by HPLC assay giving 2 (1188 g, 3.411mol, 95% yield). The resulting solution was passed through an in-linefilter (10 micron) to the reaction vessel (50 L size) for the next stepof reaction. The line was rinsed with MeCN (1-2 L) so that the totalvolume reached to about 24 L. The solution was ready for the nextreductive amination step without further purification. The mixture inCH₃CN should be protected from atmospheric moisture.

Step 2: Synthesis of9-((3aR,4R,6R,6aR)-6-(((3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-purin-6-amine(4)

The materials employed for Step 2 are as follows:

MW Grade and Reagents (density) Amount Mol. Equiv. Comment Compound 2348.4 1188 g 3.411 1.0 In MeCN and HOAc solution (24 L) from step 1Acetic acid 60.05 390 mL 6.822 2.0 ACS (1.049) Compound 3 306.83 1047 g3.411 1.0 Use test Sodium 211.94 1446 g 6.822 1.9 95% triacetoxyborohydride (STAB) Work-up Water 6 L Chromasolv MTBE 8.5 L ACS, ≥99.0%Methanol ~15 L ACS, ≥99.9% 3N NaOH* ~10 L ACS 5% NaHCO₃ 4 L ≥99.7%*Preparation: NaOH (1200 g, pellets, Fisher Scientific, Lot# 093309) wasdiluted with Chromasolv water (Sigma-Aldrich, Lot# SHBB2917V) to 10 L togive 3N NaOH.

To a 50 L 3-neck jacketed vessel with a mechanical stirrer, athermocouple, and a N2 inlet were charged 2 (1188 g, 3.411 mol) in MeCNsolution (total volume: 24 L) at room temperature (see step 1). Aceticacid (390 mL, 2.0 eq) and 3 (628 g, 0.6 eq) were added at roomtemperature under nitrogen. Since the reaction was water sensitive, thereaction mixture in CH₃CN should be protected from the moisture. Thereaction mixture as slurry was heated to 55° C., STAB (145 g, 0.19 eq)was added over 1-2 min. The remaining 3 was added in the 3 portions over4 h (209 g, 105 g, 105 g); and the remaining STAB was added over 9portions (145 g×9) over 5 h. After the additions, the resulting slurrywas stirred at 55° C. for 14-16 h. At this stage the conversion shouldbe 98 A % (“A %” refers to area percentage or are % of total area underthe peak by HPLC) or higher (relative to API-1, i.e., 2) monitored byHPLC. The LC ratio at the end of reaction: conversion by area % was >98%by HPLC, the ratio of EP-API-2-mix (i.e., 4) to RSM-2-OH (i.e., sideproduct A) was at the range of 8-9:1.

The reaction mixture was cooled to room temperature, water (6 L) wasadded over 1-2 min with stirring. The addition of water to the batch inMeCN solution was endothermic, the temperature was dropped from 25° C.to around 15° C. after the water addition. The batch was warmed to roomtemperature, the bottom aqueous layer was removed. There was no productloss in the aqueous layer. The product in MeCN solution was stable atambient temperature for up to a week, which was confirmed by HPLCanalysis. The resulting product in MeCN solution obtained was analyzedby HPLC assay, giving 4 (1.7 kg, 2.83 mol, 83% assay yield). The organiclayer was concentrated to remove most of MeCN. MTBE (8.5 L) and MeOH(1.7 L) was added. The resulting solution was cooled to 5-10° C. 3N NaOH(˜10 L) was added slowly with stirring to adjust aqueous layer pH from 6to 10, while the mixture temperature was maintained at 25-30° C. Whenthe aqueous layer reached to pH 10, the stirring was stopped and thelayers were separated. The aqueous layer was removed, and the organiclayer was washed 5% NaHCO₃(4 L). The aqueous layer was removed again,the used aqueous layer pH should be 9. The product loss in combinedaqueous layers should be less than 1.5%. The organic layer wasconcentrated, flushed with MeOH (4 L×2) to remove all MTBE. Theresulting thick oil was diluted with MeOH to about 8.5 L as a clearlight brown solution. The solution was ready for the next step directlywithout further purification.

Further note that the reason for the portion wise addition of 3 and STABwas to maintain the concentration of these two components at relativelylow concentration in the reaction mixture, which would minimize theformation of the side product A by direct 3 reduction by STAB.

A complex formed during the reductive amination reaction, which wascorresponding to several late eluting peaks by HPLC analysis. Thesepeaks were confirmed to be a complex of 4 mixture anddiacetoxyborohydride by LC/MS (B, see the structure below). This complexwas converted to the product upon treating with base (LC sample wastreated with NH₄OH to pH 10, and the complex peaks disappeared). Most ofcomplex was broken back to the product after overnight heating (55° C.)and the remaining residual amounts of complex would be broken down tothe desired product during the basic aqueous workup.

Step 3: Synthesis of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-(((3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol(5)

The materials employed for Step 3 are as follows:

MW Grade and Reagents (density) Amount Mol. Equiv. Comment Compound 4602.77 851 g 1.412 1.0 As solution in MeOH (1979 g, 43 wt %) 6N HCl*1.18 L 7.06 5.0 37%, ACS Solvents Methanol 2.9 L ACS, ≥99.8% Work-upMTBE 4.5 L ACS 3N NaOH** 2.4 L 7.2 5.1 ACS Sat'd 500 mL ACS NaHCO₃***Acetonitrile 6 L Chromasolv Acetonitrile/ 6 L water (3:1)*****Preparation: HCl (590 mL, 37%) was mixed with Chromasolv water(Sigma-Aldrich, Lot# SHBB2917V) to 1.18 L to give 6N HCl **Preparation:NaOH (1200 g, pellets, Fisher Scientific, Lot# 093309) was diluted withChromasolv water (Sigma-Aldrich, Lot# SHBB2917V) to 10 L to give 3NNaOH. ***Preparation: NaHCO₃ (Solid powder, 150 g) was mixed with water(1 L, Sigma-Aldrich, Lot# SHBB2917V) to give saturated aqueous sodiumbicarbonate solution. ****Preparation: MeCN (4.5 L, Sigma-Aldrich, Lot#HSBB0358V) and water (1.5 L, Sigma-Aldrich, Chromasolv, Lot# SHBB2917V)were premixed before use.

To a 10-L jacketed vessel with a mechanical stirrer, a thermocouple, anda N2 inlet were charged 4 (1979 g, 43%, 1.412 mol), MeOH (2.9 L), and 6NHCl (1.18 L, 5 eq). The resulting solution was heated at 45° C. for 7-9h and ambient temperature for 12-16 h (overnight). The reaction wasalmost complete after stirring at 45° C. for 7-9 h, the overnight agingwas just for the convenience. It gave the same result that the reactionwas stirred at 40° C. for 16 h. The reaction gave a full conversion atthis stage.

The reaction mixture was cooled to 5-10° C., 3N NaOH (1 L) was addedslowly keeping the temperature at the range of 25-30° C. The aqueouslayer pH should be at the range of 3-4. MTBE (3 L) was added withstirring. 3N NaOH (˜1.4 L) was added slowly with stirring keeping thetemperature at the same range of 25-30° C. The target pH should be 10.The last 10% (˜240 mL) of 3N NaOH addition should be very slow, so thatthe aqueous layer pH would be controlled to 10 without difficulty.Saturated aqueous NaHCO₃ (500 mL) was added with stirring. The aqueouslayer pH should be around 9. The layers were separated, the aqueouslayer was extracted with MTBE (1.5 L) and MeOH (375 mL) once. Thecombined organic layers were concentrated to about 1.5 L, theconcentrated residue was flushed with MeCN (2.0 L×3) to remove all MTBEand MeOH. Caution should be taken, that foaming or bumping was possibleduring the concentration. To reduce such possible foaming or bumpingbatch temperature should be kept low (<25-30° C.) during theconcentration. The resulting sticky residue was mixed with 3:1MeCN/water (4.0 L) and warmed to 45° C., obtaining a clear solution. Theassay yield of this solution (4.95 Kg, 14.56 wt %) was 91.5% as 5 (726g, 1.29 mol). The solution was transferred to a 20 L jacketed vessel viaan inline filter (Polycap 36 TC, 1.0 micron) to remove all fibers anddusts. The line was rinsed with 3:1 MeCN/water (1-2 L). The solution inthe vessel was ready for the crystallization without any furtherpurification.

Step 4: Crystallization to Isolate Pure EP-1

MW Grade and Reagents (density) Amount Mol. Equiv. Comment EP-API-mix562.71 726 g 1.29 1.0 As solution in (5) 3:1 MeCN/ water (4.95 kg, 14.45wt %) Solvents Acetonitrile* 12 L Chromasolv Water* 4 L Chromasolv*Acetonitrile and water were premixed in 3:1 ratio by volume before use.

1^(st) Recrystallization: To a 20-L jacketed vessel equipped with amechanical stirrer, a thermocouple, a N2 inlet, and with 5 (4.95 Kg,14.45 wt %, 1.29 mol) in 3:1 MeCN/water solution were charged additional3:1 MeCN/water (3˜4 L). The total volume of the batch was about 8 L andthe batch temperature was kept around 30° C. The reaction mixtureappeared as a clear light brown-yellow solution at 30° C. at this stage.If any solid was precipitated out, the mixture should be warmed to about45° C. to dissolve all and cooled back to 30° C. EP-1 solid seeds (250mg, >99.5 A %, cis/trans=99.5:0.5) was added at 30° C. with stirring.White thin slurry was generated within 30 min, the mixture was stirredat 25-30° C. for 1 h. The resulting white slurry turned thickergradually. The slurry was heated to 75° C. and stirred at the sametemperature for 1-2 h. The slurry was cooled slowly back to 30° C. over4-5 h, and stirred at the same temperature for an additional 12-16 h.The mixture was cooled to room temperature. After being stirred at thesame temperature for 2-3 h, the slurry was filtered through coarseporosity sintered glass funnel. The wet cake was washed with 3:1MeCN/water (1.5 L×2). The solid was dried in air at room temperaturewith a vacuum suction for 2-3 h to remove most of solvent. A filterpaper was covered above funnel to protect from the dust from air. Theisolated yield of this stage was about 68-70%, the purity of the solidwas typically >99 A % and 97:3 ratio of EPZ-5676/5677 (EPZ-5677, thetrans-isomer, was rejected mostly in mother liquor and during the wetcake wash). The product loss as EP-API-mix in mother liquor was 160 g,and 17 g in combined washes.

2^(nd) Recrystallization: The partially dried solid (654 g) wastransferred back to the cleaned 20 L vessel, 3:1 MeCN/water (5.5 L) wascharged. The resulting slurry was heated to 75° C. and stirred at thesame temperature for 1-2 h. The mixture was cooled slowly to 30° C. over6 h, and stirred at the same temperature for an additional 12-16 h. Themixture was cooled to room temperature. After being stirred at the sametemperature for 2-3 h, the slurry was filtered through coarse porositysintered glass funnel (medium porosity should be fine). The wet cake waswashed with 3:1 MeCN/water (1 L×2). The solid was dried in air at roomtemperature with a vacuum suction for 20-30 h to remove solvent. Afilter paper was covered above funnel to protect from the dust from air.The solids were occasionally turned over to speed up the drying process.When the weight of batch remained as constant it was considered to bedry. EPZ-5676 trihydrate was obtained (537 g, >99 A %, ratio ofEPZ-5676/5677=99.2:0.8, 66% over two crystallizations). The product lossas EP-API-mix was 19 g, and 3.5 g in combined washes.

Additional or Alternative Recrystallization: EPZ-5676/5677 (12.0 g,cis:97.07 A %, trans:2.04 A %) was mixed with 9:1 isopropyl alcohol(i.e., IPA)/H₂O (70 mL) and heated to 65° C. until dissolution. Thesolution was filtered through a fine porosity sintered glass funnel. Theflask and funnel were rinsed with 9:1 IPA/H₂O (10 mL). The filtrate washeated to 45° C. and seeded with a seed crystal of EPZ-5676 trihydrate(600 mg, >99.5 A %, cis/trans=99.5:0.5). The resulting thin slurry wasstirred at 45° C. for 2 h, and DI water (64 mL) was added via a syringepump over 12 h. The mixture was aged at 45° C. for 5 h, cooled linearlyto 15° C. over 2 h. The product was isolated by filtration and washedwith 1:1 IPA-water (2×20 mL) followed by drying in vacuo at 40° C. toconstant weight. EPZ-5676 trihydrate was obtained (11.89 g, 99% yielduncorrected, cis: 98.3 A %, trans:1.23 A %).

Example 2: Synthesis of5-tert-Butyl-2-[2-(3-oxocyclobutyl)ethyl]-1H-1,3-benzodiazol-1-iumchloride (3)

Compound 3 was prepared as described below.

Step 1: Synthesis of Pent-4-enoic acid benzyl ester (7)

Benzyl bromide (7.19 g, 42.04 mmol) was added to a solution of4-pentenoic acid (6) (5.05 g, 50.45 mmol, 1.2 eq.) in acetone (75 ml) atRT under N2. Anhydrous potassium carbonate (29.05 g, 210.19 mmol, 5.0eq.) and tetrabutylammonium iodide (0.776 g, 2.102 mmol, 0.05 eq.) wereadded and the resulting suspension was stirred over 2 days. LCMSanalysis showed mainly product.

The solid was filtered and washed with acetone. The organic solventevaporated and the residue was dissolved in EtOAc, washed with 2M HCl,sat NaHCO₃, brine, dried over Na₂SO₄, filtered and concentrated to yield7.46 g (92% yield at 99% purity) of 7 as a colorless oil. LCMS analysis(on MS19) and NMR analysis show clean product, no further purificationwas required. ¹H NMR (500 MHz, CHLOROFORM-d) δ ppm 7.31-7.42 (5H, m),5.84 (1H, ddt, J=16.98, 10.44, 6.23 Hz), 5.14 (2H, s), 4.99-5.10 (2H,m), 2.45-2.52 (2H, m), 2.37-2.45 (2H, m).

Step 2: Synthesis of 3-(2,2-Dichloro-3-oxo-cyclobutyl)-propionic acidbenzyl ester (8)

7 (7.46 g, 39.21 mmol) and zinc-copper couple (7.125 g, 98.03 mmol, 2.5eq.) in diethyl ether (128 ml) and 1,2-dimethoxyethane (19 ml) wastreated dropwise with trichloroacetyl chloride (17.83 g, 98.03 mmol, 2.5eq.). The mixture was stirred at 50° C. for 3 days. The mixture reactionwas cooled to RT, celite (˜10 g) was added and mixture stirred for ˜5min. then filtered through a plug of celite. The solid/celite werewashed with TBME (3×100 ml). The combined organic were washed with water(3×150 ml), NaHCO₃sat sol (2×150 ml), brine (100 ml), dried over Na₂SO₄,filtered and concentrated to yield a brown oil.

The brown oil was stirred with 50 ml of heptane for 10-15 min, stirringstopped and the heptane layer was removed. This was repeated until oilturn into solid (˜350-450 ml of Heptane used). The combined heptanelayers were concentrated to yield 11.29 g (96% yield at 100% purity) of8 as a yellow oil. NMR analysis shows clean product, no furtherpurification was required. ¹H NMR (500 MHz, CHLOROFORM-d) δ ppm7.32-7.43 (5H, m), 5.12-5.21 (2H, m), 3.28-3.39 (1H, m), 2.91-3.06 (2H,m), 2.49-2.65 (2H, m), 2.18-2.28 (1H, m), 2.00-2.10 (1H, m).

Step 3: Synthesis of 3-(3-Oxo-cyclobutyl)-propionic acid benzyl ester(9)

A solution of benzyl 3-(2,2-dichloro-3-oxocyclobutyl)propanoate (8)(11.29 g, 37.49 mmol) in AcOH (100 ml) was treated in small portionswith zinc powder (12.26 g, 187.44 mmol, 5 eq.) at RT. After addition,the reaction mixture was stirred at 80° C. for 2 h. LCMS analysis after2 h shows complete consumption of starting material. The reaction wascooled to RT, diluted with TBME (˜100 ml), filtered and concentrated invacuo. Heptane (250 ml) was added to remove most of the acetic acidazeotropically. Water (100 ml) was added to the resultant viscous liquidand the mixture was extracted with EtOAc (100 ml×2). The combinedorganic phase was washed with saturated NaHCO₃(100 ml×1), brine, driedover Na₂SO₄, filtered and concentrated to yield 8.12 g (93% yieldat >95% purity by NMR) of 9 as a clear yellow oil. LCMS analysis on MS19shows 92% purity. ¹H NMR (500 MHz, CHLOROFORM-d) δ ppm 7.30-7.43 (5H,m), 5.14 (2H, s), 3.08-3.21 (2H, m), 2.64-2.76 (2H, m), 2.34-2.48 (3H,m), 1.96 (2H, q, J=7.62 Hz):

Step 4: Synthesis of 3-(3-Oxo-cyclobutyl)-propionic acid (10)

A solution of 3-(3-Oxo-cyclobutyl)-propionic acid benzyl ester (9) (8.12g, 34.96 mmol) in ethyl acetate (80 ml) was purged 3× with N2 before 10%Pd/C (800 mg, 0.077 mmol, ˜2 mol %) was added. The reaction mixture waspurged again 3× with N2 then twice with H₂ before leaving the reactionunder an atmosphere of H₂. The reaction was monitored by LCMS until nomore sign of starting material was observed (˜10 h). The reaction waspurged with 3 times N2, filtered through celite and Pd/C was washed 3×with ˜25 ml of EtOAc. The combined organic were concentrated to yield4.94 g (99%) of 10 as a light yellow oil. NMR analysis shows cleanproduct, no further purification was required. ¹H NMR (500 MHz,CHLOROFORM-d) δ ppm 11.46 (1H, br. s.), 3.09-3.29 (2H, m), 2.63-2.80(2H, m), 2.35-2.53 (3H, m), 1.95 (2H, q, J=7.57 Hz).

After overnight under high vacuum, 10 solidified as a white wax with am.p. of 43° C.

Step 5: Synthesis ofN-(4-tert-Butyl-2-nitrophenyl)-3-(3-oxocyclobutyl)propanamide (12)

3-(3-Oxocyclobutyl)propanoic acid (10) (1.610 g, 11.3 mmol) and4-tert-butyl-2-nitroaniline (11) (2.000 g, 10.3 mmol) were dissolved in1,4-dioxane (20 ml) and pyridine (2.6 ml, 30.9 mmol) and T₃P (50%solution in EtOAc) (9.1 ml, 15.5 mmol) was added at r.t. The reactionwas heated to 100° C. and left for 7 hrs. The reaction was cooled tor.t., diluted with EtOAc (20 ml) and washed with 2M NaOH (2×20 ml), 2MHCl (20 ml), brine (20 ml), dried over MgSO₄ and concentrated in vacuoto give the crude product. The product was purified by silica flashcolumn chromatography using between 100% heptanes to 40% EtOAc:60%heptanes as eluent to give 12 as a yellow oil (2.776 g, 85%): MS (ESI⁺)for C₁₇H₂₂N₂O₄ m/z 319.25 [M+H]+, 341.00 [M+Na]+; LC purity 99% (UV)(ret. time, 2.11 min); H NMR (500 MHz, CDCl₃) δ 10.28 (s, 1H), 8.66 (d,J=8.9 Hz, 1H), 8.20 (d, J=2.3 Hz, 1H), 7.70 (dd, J=8.9, 2.3 Hz, 1H),3.30-3.12 (m, 2H), 2.86-2.69 (m, 2H), 2.50 (ddd, J=30.3, 14.9, 7.6 Hz,3H), 2.07 (q, J=7.6 Hz, 2H), 1.34 (s, 9H).

Step 6: Synthesis of5-tert-Butyl-2-[2-(3-oxocyclobutyl)ethyl]-1H-1,3-benzodiazol-1-iumchloride (3)

N-(4-tert-Butyl-2-nitrophenyl)-3-(3-oxocyclobutyl)propanamide (12)(2.776 g, 8.72 mmol) was dissolved in AcOH (55 ml) and iron powder wasadded (2.922 g, 52.3 mmol) at r.t. The reaction was heated to 80° C. andleft for 1 hr. The reaction was cooled to r.t. and the mixture filteredthrough GF (glass fibre) filter paper under suction and the solid waswashed with EtOAc. The solvents were removed in vacuo and the residuewas dissolved in DCM (50 ml) and sat. Na₂CO₃ solution (100 ml) was addeduntil the mixture was no longer acidic. The mixture was filtered throughCelite under suction and the plug washed with DCM. The layers wereseparated and the aqueous layer was extracted with DCM (2×50 ml). Thecombined organic layers were dried over MgSO₄, filtered and concentratedin vacuo to give the crude product. The product was salted by dissolvingthe residue in DCM (10 ml) and adding 2M HCl in ether (10 ml). Afterabout 30 seconds of swirling the solvent a white precipitate formed. Theprecipitate was filtered under suction, washed with ether and driedunder vacuum at 50° C. for 2 hrs to give the pure 3, which was pureenough for use without subsequent purification, as a white powder (2.135g, 80%): MS (ESI⁺) for C₁₇H₂₂N₂O m/z 271.45 [M+H]+, 293.20 [M+Na]+; LCpurity 97% (UV) (ret. time, 1.42 min); ¹H NMR (500 MHz, CDCl₃) δ 7.67(ddd, J=9.2, 6.4, 2.3 Hz, 3H), 3.25-3.15 (m, 4H), 2.86-2.68 (m, 2H),2.57-2.35 (m, 1H), 2.19 (dd, J=15.6, 7.7 Hz, 2H), 1.40 (s, 9H).

Example 2A: Synthesis of5-tert-Butyl-2-[2-(3-oxocyclobutyl)ethyl]-1H-1,3-benzodiazol-1-iumchloride (3) Step 1: Synthesis of benzyl 4-pentenoate (7)

A 20-L, jacketed reactor equipped with a mechanical stirrer, refluxcondenser, temperature probe and a N₂ inlet was charged with K3P04 (2.23kg, 10.5 mol, 0.7 eq.), potassium iodide (373.1 g, 2.2 mol, 0.15 eq.),Bu₄NBr (241.2 g, 0.7 mol, 0.05 eq.), water (4.5 L), and toluene (4.5 L).To the stirring mixture at 20° C. was slowly added 4-pentenoic acid (6)(1.5 kg, 15.0 mol), followed by BnCl (2.1 kg, 17.0 mol, 1.13 eq.). Theresulting mixture was heated to 62-65° C. and aged for 22 h. The batchwas assayed for completion by HPLC (210 nm) and Gas Chromatography (GC).The desired benzyl 4-pentenoate (7) was observed as the major product byboth GC and HPLC, and no 4-pentenoic acid was observed by GC.

GC sampling procedure: An aliquot from the batch containing both theaqueous layer and the organic layer was first quenched in ca. 0.5 mL of10% aq citric acid (resulting pH=4). The sample was diluted with ca. 0.5mL of MeOH to a clear solution. The resultant sample was analyzed by GCfor 4-pentenoic acid.

The batch was treated with triethylamine (454.8 g, 4.5 mol, 0.3 eq.),and the resulting reaction mixture stirred at 62-65° C. for 22 h. Thebatch was assayed by HPLC and no BnCl or BnI was observed. The stirringwas stopped, and the settled aqueous layer was removed from the bottomof the reactor. The remaining organic layer was washed with H₂O (4.5 L)at 65° C., and the settled aqueous layer was discarded. The organiclayer was cooled to 25° C., dried over Na₂SO₄ (1.5 kg), filtered througha pad of Solka Floc 40 (400 g, soaked in toluene), and concentrated invacuo to afford 4.12 kg of a light brown liquid (lot #356-78-5, 70.9 wt% in toluene by ¹H NMR with internal standard, 102% yield). HPLCanalysis of the liquid showed 99.5 A % benzyl 4-pentenoate (excludingtoluene). GC analysis showed 99.3 A % benzyl 4-pentenoate with no4-pentenoic acid.

Step 2: Synthesis of 3-(2,2-Dichloro-3-oxo-cyclobutyl)-propionic acidbenzyl ester (8)

Compound 7 (71 wt % in toluene; 1.07 assay kg; 5.64 moles) was chargedto a 10 L cylindrical vessel with dioxane (6 L). The resulting solutionwas treated with Zn—Cu couple (1400 g; 3.8 eq.) at 45° C. followed bytrichloroacetyl chloride (1.50 L; 13.45 moles; 2.4 eq.) over 5 h whilemaintaining a reaction temperature between 50-80° C. After addition wascomplete, the batch stirred at 60-65° C. for 1 h, after which time GCshowed <0.1% 7 remaining. The batch was cooled to 20° C. and stirredovernight. The batch was filtered through Solka-Floc (800 g) and thefilter cake was washed with dioxane (4 L). The combined filtrate wasconcentrated in vacuo at 40-45° C. until no more volatiles distilled togive crude 8 (3.57 kg, 5.64 mol theory, GC 86 area %).

Step 3. Synthesis of 3-(3-Oxo-cyclobutyl)-propionic acid benzyl ester(9)

To a stirred suspension of 8 (1.70 assay kg 5.64 mol) in glacial aceticacid (4.9 L) was added Zn dust (6-9μ, Alfa Aesar) (1.60 kg, 4.3 eq.) inportions over 6 h (40-90° C.). The batch was stirred at 60° C. for 0.5 hand then at ambient temperature overnight. GC analysis showed thereaction was complete (>99.0% conversion). The batch was cooled to 25°C. over several hours then stirred overnight. The batch was filteredthrough Solka-Floc (400 g) and the filter cake washed with ethyl acetate(4×2 L). The combined filtrate was concentrated under reduced pressure,and the residue partitioned between ethyl acetate (6 L) and water (6 L).The aqueous layer separated, and the organic layer washed with 1M KHPO₄(2×3 L), and then water (2 L). The organic phase was collected, driedover Na₂SO₄, and stored at 5° C.

Purification: Three batches were combined and the volatiles evaporatedto give 4.76 kg crude 9 (55 wt %, 2.62 assay kg), an 83% yield from 6.The material was purified in 3 runs as follows:

1.8 kg of crude 9 (1.0 assay kg) was eluted through a 5 kg pre-packedsilica cartridge, eluting with 9:1 hexanes/ethyl acetate. The rich cutswere pooled (64 L total) and evaporated to afford purified 9 (0.98 assaykg, 97.7 A %) in 98% recovery. Overall, the three purifications provided2.44 kg of material (96 A %; @ 90 wt %=2.20 assay kg) which represents a69% corrected yield from 7.

Step 4. Synthesis of dicyclohexylammonium 3-(3-oxocyclobutyl)propanoate(10B)

Silica gel purified 9 (1.20 kg, ˜85 wt %; 4.39 assay moles) wasdissolved in isopropyl acetate (i.e., IPAc, 6 L) and toluene (2 L) in a20-L high pressure vessel with an overhead stirrer. The 10% Pd/Ccatalyst (120 g, 50% wet) was added, and the resulting mixture waspurged by alternating vacuum and nitrogen cycles (3×), followed byvacuum and hydrogen gas (3×). The mixture was reacted with hydrogen gas(60 psi) at 20-25° C. for 20 h giving >99% conversion by ¹H NMR. Thereaction mixture was removed from the reactor which was rinsed with IPAc(800 mL) and stored separately. The batch was filtered through a mediumporosity sintered glass funnel, and washed the IPAc vessel rinse. Thefiltrate was combined with a second hydrogenolysis batch (1067 g; ˜85 wt%; 3.90 assay moles) for the dicyclohexylammonium (DCHA) salt formationwithout further purification.

The combined filtrate containing the free base (estimated 7.82 assaymoles) as a clear, colorless solution (˜18 L) was transferred to a 30 Ljacketed vessel equipped with an overhead stirrer, temperature probe andnitrogen inlet. DCHA (1.70 kg, 1.2 eq.) was added over 40 min. The batchturned cloudy after about ⅔ of DCHA was added, and then seed with a seedcrystal of compound 10B (1 g) was added. There was a moderate exothermobserved during the addition of DCHA as the internal temperature rosefrom 17° C. to 28° C. After the addition, the resulting slurry wasstirred at 20-25° C. for 18 h. The product was isolated by filtrationand the wet cake was washed with IPAc (2×3 L). The product was dried invacuo (45-55° C.) with a nitrogen sweep for three days to achieveconstant weight. Compound 10B (2.286 kg, >99 wt %) was isolated in 85%yield as a white solid (KF: 0.11 wt %). No residual solvent wasdetectable by ¹H NMR as shown in FIG. 192 . The product lost to thecombined mother liquor and washes was 271 g (10.6%).

Step 5. Synthesis ofN-(4-tert-Butyl-2-nitrophenyl)-3-(3-oxocyclobutyl)propanamide (12)

To a 30 L jacketed vessel with equipped with an overhead stirrer,temperature probe and nitrogen inlet, was charged 10B (1.1 kg),1,4-dioxane (11 L), and DMF (2.63 mL). The resulting mixture was cooledto 12° C. and oxalyl chloride (299.4 mL, 1.02 eq.) was added at 12-17°C. over 35 min, followed by aging at 18-20° C. for 18 h. The conversionof 10B to the corresponding acid chloride 10C was monitored by quenchinga reaction sample with excess Me₂NH. Specifically, 50 μL of reactionsample was quenched with access amount of Me₂NH (0.16 mL, 2 M in THF) inTHF (0.3 mL). 20 μL of such quenched mixture was diluted with 1:1MeCN/H₂O to 1.0 mL and analyzed by LC, which indicated >99.5 A %conversion. A solution of 4-t-butyl-2-nitroaniline (i.e., compound 11,628 g) in 1,4-dioxane (1.88 L) was added to the reaction mixture over 60min at 15-20° C. The resulting orange-yellow slurry was stirred at 20°C. for 1 h, and slowly warmed to 35-40° C. over 4 h and aged for 1 h.The batch was cooled to 20° C. over 2 h and aged for 18 h givingcomplete conversion of nitroaniline to compound 12, H NMR of which isshown in FIG. 193 .

The batch was filtered to remove solid DCHA HCl salt. The wet cake waswashed with 1,4-dioxane (3×4 L). The filtrate was combined with another550 g batch and then concentrated in vacuo at 45° C., flushed with AcOH(3×2.5 L) and diluted with AcOH to ˜6.6 L. The batch was warmed to 35°C., and DI water (5.9 L) was added over 2 h. The batch was seeded with aseed crystal of compound 12 (1 g) after 2 L of water was added. Anorange slurry gradually formed, which was stirred at 30-35° C. for 3 h,and then at 18-20° C. for 14 h. The slurry was filtered and the wet cakewashed with 2:3 AcOH/H₂O (2×3.5 L). 1.59 kg of compound 12 as yellowsolid was obtained after partial drying in vacuo at 50° C. The materialgave 98.4 A %, 89 wt % (partially wet) by LC assay and in 89% isolatedyield based on the nitroaniline charge (compound 11). The product lossin mother liquor and wash was 3.3%.

Step 6. Synthesis of5-tert-Butyl-2-[2-(3-oxocyclobutyl)ethyl]-1H-1,3-benzodiazol-1-iumchloride (3)

To a 20 L jacketed vessel equipped with an overhead stirrer, temperatureprobe and nitrogen inlet was charged 12 (1.32 kg, 89 wt %; 3.69 assaymoles) and AcOH (14.1 L, 12 vol). The resulting solution was heated to45° C., and iron powder (723 g, 3.5 eq.; ˜325 mesh) was charged in 3equal portions over 1 h at 45-67° C. (addition was very exothermic). Thereaction was complete after aging at 65-75° C. for 3 h. The batch wascooled to 20° C. over 5 h and aged overnight. The resulting slurry wasfiltered, combined with the 100 g front run, and the wet cake washedwith toluene (3×3 L). The combined filtrate was concentrated, andflushed with toluene (2×2 L) to remove most of the AcOH. The crude 3free base, as a thick oil (2245 g, containing AcOH and toluene), assayedat ˜95% assay yield. There was no product trapped in the iron salt wetcake. The crude 3 free base was diluted with DCM (˜-8 L) to ˜10 L andtransferred to a 30 L vessel. The solution was neutralized by adding 10wt % aq. Na₂CO₃ (8 kg) slowly over 1 h at 20-30° C. The aqueous layer pHreached 7 after all the Na₂CO₃ solution was added. There was no productloss in the spent aqueous layer. The organic layer was then dried overNa₂SO₄ (2.5 kg), washed with DCM (2×2.5 L). Compound 3 free basesolution in DCM analyzed at 97.0 A %. The solution was then concentratedto 3.8 kg and re-analyzed by ¹H NMR.

The batch was transferred to 20 L jacketed vessel, and diluted with DCMto ˜9.1 L. The resulting solution was cooled to 0-5° C., and 4 N HCl indioxane (1.0 L) was added slowly over 1 hour at 5-10° C. After ˜50% ofthe HCl was added, the batch was seeded with a seed crystal of compound3 (˜2.5 g). Once all the HCl was added, the batch temperature was raisedto 18-20° C. The resulting slurry was aged for 17 h, and the batch wasfiltered and washed with DCM (2×3 L). The wet cake was dried in vacuo(40-45° C.) with a nitrogen sweep for two days. 1068 g of 3 was isolatedas an off-white solid in 87% yield (see HPLC, NMR and LC-MS spectra inFIGS. 194-196 ). The detailed analytical information is summarized inthe table below.

Compound 3 Analytical Data

Test Result Comment LC purity (KABS) 99.5 A % KF 0.26 wt % Chloride (wt%) by 11.05 wt % Theoretical: 11.90 ROI <0.10% Residual solvent Ethylacetate ND Isopropyl acetate ND Acetic acid 220 ppm Acetone NDDichloromethane 177 ppm Hexane 180 ppm Dioxane ND Toluene ND Mass Spec271.2 theory m/z= Pd <10 ppm* Cu <10 ppm* Zn <10 ppm* Fe 53 ppm*

Example 3: Polymorph Screening

A screening strategy using different crystallization methods indifferent solvents was applied to maximize the probabilities of findingas many crystal forms as possible. In the present study, fivecrystallization methods were utilized for polymorph screening, namelyslow evaporation, solvent-mediated phase transition, anti-solventaddition, solvent sweeping, and vapor diffusion. The starting materialused for the screening was lot EP-1 trihydrate (x is 3). This materialwas found to be Form B as indicated by solid state characterization.

Slow Evaporation

Slow evaporation experiments were performed in 32 solvents by dissolving˜15-20 mg of EP-1 trihydrate (x is 3) (Form B) in 0.4-2.0 ml of solventin a 3-ml vial; the resulting clear solutions were left with the capsoff and subjected to slow evaporation to produce precipitation. Thesolids were isolated for X-ray Powder Diffraction (XRPD) analysis andthe results are summarized in Table 1. As shown in Table 1, either FormB or amorphous phase was produced in all tested solvents.

TABLE 1 Slow Evaporation Solid Solid Solvent Obtained NB-Ref SolventObtained benzonitrile Type B 802401-37-A17 1:4 (v:v) EtOH/hexaneamorphous trifluoroethanol amorphous 802401-37-A18 1:4 (v:v) acetone/H₂OForm B THF^(a) Type B 802401-37-A19 1:4 (v:v) ACN^(h)/H₂O Form BEtOAc^(b) Type B 802401-37-A20 1:4 (v:v) amorphous acetone/MTBE 1,2-Type B 802401-37-A21 1:4 (v:v) IPAC/MTBE Form B dichloroethane CH₂Cl₂amorphous 802401-37-A22 1:4 (v:v) ACN/MTBE Form B anisole amorphous802401-37-A23 1:4 (v:v) THF/H₂O Form B IPA^(c) amorphous 802401-38-A11:4 (v:v) IPA/H₂O Form B Me—THF^(d) amorphous 802401-38-A2 1:4 (v:v)IPA/hexane Form B toluene Type B 802401-38-A3 1:4 (v:v) amorphousacetone/hexane IPAC^(e) Type B 802401-38-A4 1:4 (v:v) THF/hexane Form Bcyclohexanol Type B 802401-38-A5 MTBE Form B acetic acid amorphous802401-38-A6 glycol Form B cyclohexane amorphous 802401-38-A71,2-dimethoxyethane amorphous 1:4 (v:v) Type B 802401-39-A8 1:4 (v:v)EtOH/MTBE Form B EtOH^(f)/H₂O 1:4(v:v) Type B 802401-39-A9 1:4 (v:v)Form B THF/MTBE^(g) MeOH^(i)/MTBE ^(a)THF: tetrahydrofuran; ^(b)EtOAc:ethyl acetate; ^(c)IPA: isopropyl alcohol; ^(d)Me—THF:2-methyltetrahydrofuran; ^(e)IPAC: isopropyl acetate; ^(f)EtOH: ethanol;^(g)MTBE: methyl t-butyl ether; ^(h)ACN: acetonitrile; ^(i)MeOH:methanol.Solvent-Mediated Phase Transition

Solvent-Mediated phase transition experiments were conducted in twelvesolvents by suspending ˜20-30 mg EP-1 trihydrate (x is 3) (Form B) in0.5-1.0 ml of solvent at both RT and 50° C. After the suspension wasstirred for 3 days, the remaining solids were isolated for XRPDanalysis. The results summarized in Table 2 indicate that either Type Bor amorphous phase was generated.

TABLE 2 Solvent-Mediated Phase Transition Remaining Solvent Temp. TimeSolid toluene RT* 3 days Form B CHCl₃ RT 3 days Form B ACN RT 3 daysForm B IPAC RT 3 days Form B 1:4 (v:v) dioxane/heptane RT 3 days Form B1:4 (v:v) acetone/heptane RT 3 days Form B 1:4 (v:v) THF/heptane RT 3days Form B 1:4 (v:v) Me—THF/MTBE RT 3 days Form B 1:4 (v:v) THF/H₂O RT3 days Form B 1:4 (v:v) IPA/H₂O RT 3 days Form B 1:4 (v:v) EtOH/MTBE RT3 days amorphous 1:4 (v:v) MeOH/MTBE RT 3 days Form B toluene 50° C. 3days Form B CHCl₃ 50° C. 3 days Form B ACN 50° C. 3 days amorphous IPAC50° C. 3 days Form B 1:4 (v:v) dioxane/heptane 50° C. 3 days Form B 1:4(v:v) acetone/heptane 50° C. 3 days Form B 1:4 (v:v) THF/heptane 50° C.3 days Form B 1:4 (v:v) Me—THF/MTBE 50° C. 3 days Form B 1:4 (v:v)THF/H₂O 50° C. 3 days Form B 1:4 (v:v) IPA/H₂O 50° C. 3 days Form B *RT:Room Temperature (25 ± 3° C.)Anti-Solvent Addition

A total of twenty anti-solvent addition experiments were carried out bydissolving ˜20 mg EP-1 trihydrate (x is 3) (Form B) in 0.3-3.0 ml goodsolvent to obtain saturated solution. 1.0-3.0 ml anti-solvent was addedto the saturated solution to precipitate out solids. XRPD was then usedto analyze the precipitated solids and the results are summarized inTable 3. Either Form B or amorphous phase was formed.

TABLE 3 Anti-Solvent Addition Solid Solvent Anti-solvent TemperatureObtained acetone hexane RT* amorphous THF hexane RT Form B IPA hexane RTamorphous dioxane hexane RT Form B acetone heptane RT amorphous THFheptane RT Form B IPA heptane RT Form B dioxane heptane RT Form B MeOHH₂O RT Form B Acetone H₂O RT Form B ACN H₂O RT Form B THF H₂O RT Form BDioxane H₂O RT Form B IPA H₂O RT Form B EtOH H₂O RT Form B acetic acidH₂O RT Form B acetone MTBE^(f) RT amorphous ACN MTBE^(f) RT Noprecipitation observed THF MTBE^(f) RT amorphous acetic acid MTBE^(f) RTamorphous *RT: Room Temperature (25 ± 3° C.)Vapor Sweeping

Vapor sweeping experiments in eleven solvents at RT were conducted byplacing ˜10 mg amorphous EP-1 trihydrate (x is 3) (Form B) into a 1-mlvial which was put inside a 20-ml vial filled with ˜5 ml volatilesolvents. The bigger vials were sealed with a cap and kept at roomtemperature for 7 days allowing sufficient time for organic vapor tointeract with the solids. The solids were analyzed at the end of theexperiment and the results are listed in Table 4. Either Form B oramorphous phase was generated.

TABLE 4 Results from Vapor Sweeping Solid NB-Ref Solvent TemperatureTime Obtained 802401-45-A1 Me—THF RT* 7 days Form B 802401-45-A2 butanolRT 7 days solid deliquesced 802401-45-A3 THF RT 7 days Form B802401-45-A4 EtOAc RT 7 days Form B 802401-45-A5 MeOH RT 7 days Form B802401-45-A6 toluene RT 7 days amorphous 802401-45-A7 acetone RT 7 daysamorphous 802401-45-A8 ACN RT 7 days Form B 802401-45-A9 hexane RT 7days amorphous 802401-45-A10 CH₂Cl₂ RT 7 days amorphous 802401-45-A11IPAC RT 7 days amorphous *RT: Room Temperature (25 ± 3° C.)Vapor Diffusion

Vapor diffusion experiments in eleven solvents at RT were conducted bydissolving ˜10 mg amorphous EP-1 trihydrate (x is 3) (Form B) in ˜0.5 mlappropriate solvent to obtain a clear solution in a 3-ml vial, thissolution was placed inside a 20-ml vial filled with ˜5 ml volatilesolvents. The larger vials were sealed with a cap and kept at roomtemperature for 14 days, allowing sufficient time for organic vapor ofthe anti-solvent to diffuse into the solution of EP-1 trihydrate (x is3) to precipitate out solids. The solids obtained were separated andanalyzed with XRPD. The results are summarized in Table 5. Onlyamorphous phase was formed in these experiments.

TABLE 5 Results from Vapor Diffusion Experiments Starting Solid NB-RefMaterial Solvent Anti-Solvent Obtained 802401-50-A1 amorphous THFheptane amorphous 802401-50-A2 amorphous THF MTBE no precipitationobserved 802401-50-A3 amorphous THF toluene amorphous 802401-50-A4amorphous IPA heptane amorphous 802401-50-A5 amorphous IPA MTBEamorphous 802401-50-A6 amorphous IPA toluene amorphous 802401-50-A7amorphous IPA EtOAc amorphous 802401-50-A8 amorphous n-butanol heptaneamorphous 802401-50-A9 amorphous n-butanol MTBE no precipitationobserved 802401-50-A10 amorphous n-butanol toluene amorphous802401-50-A11 amorphous n-butanol EtOAc no precipitation observed

Example 4: Physical Characterization and Thermodynamic PhaseRelationships

Three crystalline forms have been observed for free base EPZ-5676: TypeA and Type B are hydrates while Type C is an anhydrate. All three solidforms have been fully characterized and their thermodynamicrelationships have been investigated.

Form A

Form A may be observed EP-1. EP-1 may be crystalline but contains acertain amount of amorphous phase as evidenced by the halo displayed inthe XRPD pattern (FIG. 1 ). The DSC curve (FIG. 2 ) of Form A exhibits adehydration endotherm at ˜80° C. (peak temperature) which is accompaniedby 5.9 wt % weight loss up to ˜150° C. in the TGA curve (FIG. 3 ). Thewater content was found to be 6.4 wt % as determined by a Mettler ToledoDL31 KF Titrator. This data confirms that Form A is a hydrate. Form A ishygroscopic as indicated by water adsorption of ˜5.8 wt % at 80% RHmeasured by dynamic vapor sorption (DVS) (FIG. 4 ). A solid isconsidered moderately hygroscopic when water uptake at 25° C./80% RH isbetween 2-15 wt % based on criteria of European Pharmacopeia. Thehysteresis in the DVS plot suggests that Form A partially converts toForm B during the experiment, although it converts back to Form A whenRH goes back down to zero, therefore no significant change in XRPDpattern was observed post DVS experiment (FIG. 5 ).

Form B

Form B of EP-1 trihydrate (x is 3) is a trihydrate as confirmed bysingle crystal analysis. The experimental XRPD pattern of Form B matcheswell with the simulated one from single crystal data (FIG. 6 ). This wasalso confirmed by DSC and TGA data (FIG. 7 and FIG. 8 , respectively).DSC data indicates Form B dehydrates at ˜132° C. (peak temperature) andTGA data displays a 8.73% weight loss up to 160° C. which is in goodagreement with the theoretical value (8.75%) of a trihydrate. Form B isslightly hygroscopic as evidenced by a weight change of ˜0.3% between 0%and 95% RH (FIG. 9 ), (data collection was programmed from 95% RH-0%RH-95% RH at a step size of 10% RH at 25° C.). XRPD results suggestedthat no form change was observed for Form B after the DVS experiment(FIG. 10 ).

Form C

Form C was observed upon heating of Form B to 120° C. and cooling downto room temperature (FIG. 11 ). Its DSC and TGA characterization dataare shown in FIG. 12 and FIG. 13 , respectively. The results suggest itis a channel hydrate.

Thermodynamic Phase Relationships

Variable temperature XRPD indicates that Form B converts to Form C uponheating to 120° C., and cooling down to RT in the presence of ambientmoisture (FIG. 14 ). Form C is an anhydrate form whose XRPD pattern isvery similar to that of Form A.

The relative stability between Form A and Form B was investigated byslurry experiments in ACN (containing less than 0.3 wt % water), ACN/H₂O(v/v, 3; 1), and H₂O at room temperature and 50° C. In general, about10-30 mg Form A was added to a 5-ml glass vial with 0.5 ml correspondingsolvents to get suspensions at room temperature and 50° C.,respectively. Then 10-30 mg Form B was added to the suspensions whichwere kept stirring for 24 h. The water activity was calculated using theUNIFAC model. The results summarized in Table 6 indicate that Form B isthermodynamically more stable than Form A at water activity equal to orgreater than 0.09 at RT and 0.08 at 50° C. as evidenced by formconversion of Form A to Form B in ACN (containing less than 0.3 wt %water), ACN/H₂O (v/v, 3:1), and H₂O (FIG. 15 and FIG. 16 ).

TABLE 6 Results of Slurry Experiments Using Form A and Form B WaterStarting Solvent Activity Form Temperature Ending Form ACN <0.09^(§) A +B RT* Form B 3:1 ACN/H₂O 0.93 A + B RT Form B H₂O 1.00 A + B RT Form BACN <0.08 A + B 50° C. Form B 3:1 ACN/H₂O 0.90 A + B 50° C. Form B H₂O1.00 A + B 50° C. Form B ^(§)ACN contains less than 0.3 wt % water. *RT:Room Temperature, 25 ± 3° C.

The relative stability between Form B and Form C was investigated as afunction of water activity at room temperature using ACN/H₂O. Ingeneral, about 10-30 mg Form B was added to a 5-ml glass vial with 0.5ml corresponding solvents to get suspensions at room temperature. Then10-30 mg Form C was added to the suspensions which were kept stirringfor 24 h. The water activity was calculated using the UNIFAC model. Theresults summarized in Table 7 indicate that Form B is thermodynamicallymore stable than Form C at water activity equal to or greater than 0.30at RT (FIG. 17 ).

TABLE 7 Results of Slurry Experiments Using Form B and Form C WaterStarting Solvent Activity Form Temperature Ending Form ACN <0.09^(§) B +C RT* B + C 98.5:1.5 ACN/H₂O 0.30 B + C RT B 97:3 ACN/H₂O 0.50 B + C RTB 3:1 ACN/H₂O 0.93 B + C RT B H₂O 1.00 B + C RT B ^(§)ACN contains lessthan 0.3 wt % water. *RT: Room Temperature, 25 ± 3° C.

To study if Form A and Form B would perform differently in stomach, thesolubilities of Form A and Form B were measured in Simulated GastricFluid (SGF, see below for SGF preparation) at 37° C. In theseexperiments, about 100 mg Form A and 50 mg Form B were weighed into20-ml glass vials, followed by addition of 4 ml SGF into each vial andthe samples were stirred at 37° C. Aliquots were taken for solubilitydetermination using HPLC after 15, 30, and 60 min, respectively. Theremaining solids after each time point were subject to XRPD analysis.The results are summarized in Table 8 and FIG. 18 which indicate thatForm A converts to Form B in SGF within 15 min, suggesting Form A andForm B will behave the same after 15 min in the stomach.

TABLE 8 Solubility of Form and Form in SGF at 37° C. 15 min 30 min 60min Start- Solu- Result- Solu- Result- Solu- Result- ing bility* ingbility ing bility ing Final Form mg/ml Solid mg/ml Solid mg/ml Solid pHType A 5.4 Type B 5.5 Type B 5.3 Type B 5.1 Type B 5.4 Type B 5.4 Type B5.6 Type B 5.3 *Solubility of Form A and Form B was both calculatedusing free base content

Based on the above results, among the three observed crystalline forms,Form B is the most thermodynamically stable form under ambientconditions. The relationships of these three crystalline forms aresummarized in FIG. 19 . An XRPD overlay for Form A, Form B, and Form Cis shown in FIG. 20 .

Example 5: Further Evaluation of Form B

Based on the physical characterization data (DSC, TGA, XRPD, and DVS)and relative thermodynamic stability, Form B was deemed most suitablefor pharmaceutical development. Thus additional characterization andpreformulation evaluation is focused on Form B.

Particle Morphology

The morphology of Form B obtained by recrystallization in MeOH/H₂O isshown in FIG. 21 . Form B grows as thin plate-like crystals in MeOH/H₂O.

Solubility

Approximately 10 mg of Form B was weighed into a 1-ml glass vialfollowed by addition of 0.5 ml relevant media. Each sample wascontinuously stirred using a magnetic stir bar at 25° C. for 24 h. Thesuspension was then filtered using a nylon membrane with a pore diameterof 0.22 μm. The filtrates were diluted for HPLC analysis and final pHwas measured. The solid residue of each sample was collected for XRPDanalysis.

Equilibrium Solubility in Aqueous Media

The solubilities of Form B in water and five different pH values at 25°C. were determined by HPLC after 24 h equilibration. Solubility data forForm B was obtained across the physiological pH by using buffersolutions from pH 2 to pH 10. The results are summarized in Table 9. Thesolubility of Form B at RT in unbuffered water was found to be 1.2μg/ml. Data in Table 9 indicated that Form B has pH-dependentsolubility, namely, Form B shows satisfactory solubilities at pH ≤4(i.e., 3.4 mg/ml at pH 2 and 9.8 mg/ml at pH 4). No crystal form changewas observed in all solubility experiments (FIG. 22 ).

TABLE 9 Solubility of Form B in Aqueous Media at RT pH pH SolubilityRemaining Buffer (initial) (final) (mg/ml) Solid Water Not 5.6 1.2 ×10⁻³ Form B measured 0.01N HCl 2.0 5.5 3.4 Form B 50 mM sodium citrate4.0 5.4 9.7 Form B 50 mM sodium citrate 6.0 6.0  0.08 Form B 50 mM Naphosphate 8.0 8.0 <LOD^(§) Form B buffer 50 mM Na carbonate 10.0 10.0<LOD  Form B buffer ^(§)LOD was determined to be 0.1 μg/mlEquilibrium Solubility in Organic Solvents

The solubility of Form B was also determined in commonly used organicsolvents (diluents or HPLC mobile phase). The solubility data summarizedin Table 10 indicates Form B is quite soluble in MeOH, EtOH, and IPA.

TABLE 10 Solubility of Form B in Organic Solvents at RT SolubilityRemaining Solvent (mg/ml) Solid Acetone 23.9 Form B Methanol >200 Nosolid obtained Ethanol >87 No solid obtained Isopropanol >129 No solidobtained Acetonitrile 1.5 Form BpKa Studies

The pKa and log P were calculated use software ADMET Predictor, Version5.5 from Simulation Plus Inc. Lancaster, Calif. The predicted major pKasof EP-1 are 12.73, 7.80, 6.00 and 3.61 (the aliphatic-OH groups wereignored by the software). The microstates of the compound in differentpKa are plotted in FIG. 23 .

Solid-State Stability

Chemical Stability: Accurately weighed ˜10 mg Form B was placed intofour 10-ml volumetric flasks. The samples were stored at 5° C. closeddish, 25° C./57% RH open dish, 40° C./75% RH open dish, and 60° C.closed dish for 7 days. The sample stored at 5° C. was used as acontrol. Assay and total related substances for each sample were checkedat the end of 7 days.

Physical Stability: Accurately weighed ˜15 mg of Form B was placed into5-ml glass vials and the samples were stored at 5° C. closed dish, 25°C./57% RH open dish, 40° C./75% RH open dish, and 60° C. closed dish for7 days. Samples were analyzed by XRPD and TGA (FIG. 24 ).

No detectable physical change and no significant degradation wereobserved after 7 days under 5° C. closed dish, 25° C./57% RH open dish,40° C./75% RH open dish and 60° C. closed dish (results summarized inTable 11). This was also confirmed by the water content determination byTGA (theoretical water content of a trihydrate is 8.75 wt %).

TABLE 11 Solid-state Stability of Form B 7 Days Chemical PhysicalConditions Area %* % Claim^(§) TGA (%) XRPD 5° C. Closed Dish 99.1100.0^(&) 8.63 Form B 25° C./57% RH Open Dish 99.0 100.7 8.66 Form B 40°C./75% RH Open Dish 99.0 101.0 8.64 Form B 60° C. Closed Dish 99.1 100.78.49 Form B *Area % = 100% − TRS %; TRS: Total related substance^(§)Claim % = Cs/Ci × 100%; Cs: Concentration of stability sample; Ci:Concentration of initial sample ^(&)The sample was used as standard forthe calculationSolution Stability

0.1 mg/ml solutions of Form B were prepared in five buffers, including0.01N HCl (pH 2), 50 mM sodium citrate (pH 4), 50 mM sodium citrate (pH6), 50 mM sodium phosphate (pH 8), and 50 mM sodium carbonate (pH 10).Methanol was added as a co-solvent to dissolve Form B in buffers (i.e.,10% (v/v) in pH 2, 4, and 6 buffers, 20% (v/v) in pH 8 buffer, and 30%(v/v) in pH 10 buffer). Each buffer solution containing Form B wasstored at 3 different temperatures: 25° C., 37° C., and 50° C. Aliquotswere taken for HPLC analysis after 1 day and 7 days. pH values were alsomeasured before and after the stability experiments.

Solution stability data are summarized in Table 12. EP-1 trihydrate (xis 3) shows good stability in solution since less than 5% degradationwas observed.

TABLE 12 Solution Stability of EP-1 trihydrate (x is 3) pH 2 24 h 7 days% % % % Temp. area* claim^(§) pH area claim pH 25° C. 98.7 97.1 2.1 98.599.1 2.1 37° C. 98.7 96.8 2.1 98.4 97.5 2.1 50° C. 98.3 97.0 2.1 98.497.0 2.1 24 h 7 days % % % % Temp. area claim pH area claim pH pH 4 25°C. 99.4 97.4 4.2 99.6 100.2 4.2 37° C. 99.4 97.6 4.1 99.5 99.0 4.1 50°C. 99.4 97.6 4.1 99.5 98.7 4.2 pH 6 25° C. 99.2 98.0 6.2 99.5 100.0 6.237° C. 99.3 97.9 6.2 99.4 99.0 6.3 50° C. 99.3 98.1 6.2 99.5 98.9 6.2 pH8 25° C. 99.2 98.0 8.2 99.5 99.8 8.2 37° C. 99.5 98.4 8.2 99.5 99.3 8.350° C. 99.5 98.0 8.3 99.5 98.8 8.3 pH 10 25° C. 99.4 98.0 10.9 99.4 99.710.9 37° C. 99.4 98.2 10.9 99.4 99.0 10.9 50° C. 99.5 97.9 11.0 99.498.9 10.9 *Area % = 100% − TRS %; TRS: Total related substances.^(§)Claim % = Cs/Ci × 100%; Cs: Concentration in stability sample; Ci:Concentration in initial samplePhotostability

Solid-State Photostability

The solid-state photostability of EP-1 trihydrate (x is 3) was assessedupon exposure to UV/Vis light according to ICH guidelines. Loss of claim%, loss of area %, and degradates were evaluated versus dark controlsamples.

Solid-State Photostability: Weighed ˜5 mg Form B into glass vials with 2vials covered with foil as the controls. Put these 2 samples with theircontrols into chamber and expose them to Vis (10 Kilo lux) for 120 hrsfollowed by UV (10 W/m²) for 20 hrs (per ICH guidelines). At the end ofVis, take 1 sample and 1 control out for analysis and the others weretaken out after both Vis and UV exposure.

Solid state photostability data are summarized in Table 13. No loss ofclaim %, area %, or degradates were detected as compared to the darkcontrol samples, indicating the bulk Form B is stable upon exposure tofull ICH photostability requirement.

TABLE 13 Solid State Photostability of EP-1 trihydrate (x is 3) - Form BVis-UV stability Vis Stability (1.2 Mil lux-hours + (1.2 Mil lux-hours)200 Watt-hours/m²) Sample % area* % claim^(&) % area* % claim^(&) Bulk99.8 101.1 99.8 99.6 Bulk control 99.7 100.0 99.8 100.0 *Area % = 100% −TRS %; TRS: Total related substances ^(&)Claim % = Cs/Ci × 100%; Cs:Concentration in stability sample; Ci: Concentration in initial sampleSolution Photostability

Solution Photostability: Weighed ˜1 mg of Form B and dissolved in 3solutions at different pHs (0.01 N HCl, 0.01 N NaOH and water). Methanolwas added as a co-solvent to dissolve Form B in solutions (i.e., 10%(v/v) methanol in 0.01 N HCl, 20% (v/v) in water and 30% (v/v) in 0.01 NNaOH), the final concentration was 0.1 mg/mL. For each solution, 1sample and 1 control covered in foil were put into the photo chamber forVis study (2 vials total for each solution). Measure the concentrationand monitor the degradation.

Solution photostability data listed in Table 14 indicated that EP-1trihydrate (x is 3) (Form B) in water and 0.01N HCl solution is stableupon exposure to UV/Vis. Precipitation was observed for all solutions in0.01N NaOH. The precipitate was thus re-dissolved using methanol andchecked with HPLC. No degradation product was observed from HPLC data(FIG. 25 ) which suggested the precipitate was EP-1 trihydrate (x is 3)instead of degradates.

TABLE 14 Solution Photostability of EP-1 trihydrate (x is 3) - Form BVis-UV Stability Vis Stability (1.2 Mil lux-hours + (1.2 Mil lux-hours)200 Watt-hours/m²) % % Final % % Final Sample area* claim^(&) pH areaclaim pH H₂O Light 99.6 98.5 7.3 99.3 94.7 7.3 H₂O Dark 100.0 100.0 7.7100.0 100.0 8.1 0.01N HCl Light 98.5 98.8 2.1 99.9 99.9 2.1 0.01N HClDark 99.4 100.0 2.1 99.9 100.0 2.1 0.01N NaOH Light 99.6 44.5 11.7 99.366.3 11.7 0.01N NaOH Dark 99.7 100.0 11.6 100.0 100.0 11.7 *Area % =100% − TRS %; TRS: Total related substances ^(&)Claim % = Cs/Ci × 100%;Cs: Concentration in stability sample; Ci: Concentration in initialsampleGeneral Methods

Starting Material

The samples used in the stability study were prepared byrecrystallization of EP-1 trihydrate (x is 3) in MeOH/H₂O.

Instruments and Methods

X-Ray Powder Diffraction

Analytical Instrument: Panalytical Empyrean. The X-ray powderdiffractogram was determined by mounting a sample of the crystallinematerial on a Si single crystal holder and spreading out the sample intoa thin layer with the aid of a microscope slide. The 20 position wascalibrated against Panalytical 640 Si powder standard. The sampleirradiated with X-rays generated by a copper long-fine focus tubeoperated at 45 kV and 40 mA with a wavelength of Kα1=1.540598 angstromsand Kα2=1.544426 angstroms (Kα2/Kα1 intensity ratio is 0.50). Thecollimated X-ray source was passed through an programmed divergence slitset at 10 mm and the reflected radiation directed through a 5.5 mmantiscatter slit. The sample was exposed for 12.7 seconds per 0.0167°2-theta increment (continuous scan mode) over the range 3 degrees to 40degrees 2-theta in theta-theta mode. The running time was 3 minutes and57 seconds. The instrument was equipped with a RTMS detector(X'Celerator). Control and data capture was by means of a Dell Optiplex780 XP operating with data collector software. Persons skilled in theart of XRPD will realise that the relative intensity of peaks can beaffected by, for example, grains above 30 microns in size andnon-unitary aspect ratios that may affect analysis of samples. Theskilled person will also realise that the position of reflections can beaffected by the precise height at which the sample sits in thediffractometer and the zero calibration of the diffractometer. Thesurface planarity of the sample may also have a small effect. Hence thediffraction pattern data presented are not to be taken as absolutevalues. The typical XRPD parameters used are listed in Table 15.

TABLE 15 Typical XRPD Parameters Parameters for Reflection Mode X-Raywavelength Cu, kα, Kα1 (Å): 1.540598, Kα2 (Å): 1.544426 Kα2/Kα1intensity ratio: 0.50 X-Ray tube setting 45 kV, 40 mA Divergence slitAutomatic Scan mode Continuous Scan range (°2TH) 2°-40° Step size (°2TH)0.0170 Scan speed (°/min) About 10

Differential Scanning Calorimetry (DSC)

Instrument: TA Q200 DSC from TA Instruments

Method: ramp from RT to desired temperature at a heating rate of 10°C./min using N2 as the purge gas, with pan crimped.

Thermogravimetic Analysis (TGA)

Instrument: TA Q500 TGA from TA Instruments

Method: ramp from RT to desired temperature at a heating rate of 10°C./min using N2 as the purge gas.

HPLC Method

Agilent 1100 HPLC was utilized and detailed chromatographic conditionsare listed in Table 16 and Table 17.

TABLE 16 Chromatographic Conditions and Parameters Conditions Column:Cosmosil 5C18-MS-II 4.6 × 250 mm Flow Rate: 1.0 ml/min. Injection Volume10 μL Detector Wavelength: 254 nm Run time: 40 min. Column Temperature:40.0° C. Post Time 5 min.

TABLE 17 Gradient of Mobile Phase Time Mobile Phase A Mobile Phase B(min) (100% Acetonitrile) (%) (0.1% NH₄OH in H₂O): (%) 0 10.0 90.0 40.070.0 30.0 40.1 10.0 90.0

Solution Preparation

Simulated Gastric Fluid (SGF) Preparation:

Weigh appropriate amount of hydrochloride (HCl) and sodium chloride(NaCl) into a 1-L flask followed by addition of 1-L deionized water. Themixture was stirred until all solids are dissolved. Check pH value witha pH-meter and adjust the pH to 1.8 with HCl (1N) or NaOH (1N).

Preparation of pH Buffers

-   -   pH 2: 0.01N HCl solution. Dilute 1 ml of 1 N HCl with H₂O up to        a total volume of 100 ml. Measure the pH and precisely adjust to        pH=2.00 using a few microliters of 1N HCl or 1N NaOH.    -   pH 4: 50 mM Na citrate buffer. Prepare a 50 mM citric acid        solution by diluting 10 ml of 1N citric acid with H₂O up to a        total volume of 200 ml. Slowly add solid NaOH under stirring to        increase the pH to 3.8-4.0 and use a few microliters of 1N NaOH        for final adjustment to pH 4.00.    -   pH 6: 50 mM Na citrate buffer. Use the same procedure as the pH        4 buffer and adjust to pH=6.00    -   pH 8: 50 mM Na phosphate buffer. Prepare a 50 mM NaH₂PO₄        solution by dissolving 6.00 g NaH₂PO₄ in 1 L of H₂O and a 50 mM        Na₂HPO₄ solution by dissolving 7.10 g in 1 L of H₂O. Mix 10 ml        of the NaH₂PO₄ solution with 140 ml of the Na₂HPO₄ solution in a        beaker. Adjust to pH 8.00 by addition of small amounts of 50 mM        NaH₂PO₄ or Na₂HPO₄ solution.    -   pH 10: 50 mM Na carbonate buffer. Prepare a 50 mM carbonate        solution by dissolving 840 mg of sodium bicarbonate (NaHCO₃) in        about 180 ml of H₂O. Slowly adjust the pH to 10.0±0.1 by        addition of 1N NaOH. Add water to reach the total volume of 200        ml and adjust to pH 10.00 with 1N NaOH.

In summary, in Examples 3-5, a polymorph screening and selection hasbeen performed for EP-1. Three crystalline forms Form A, Form B and FormC have been obtained and evaluated. The highly crystalline Form B, whichis a trihydrate, was identified to be suitable for pharmaceuticaldevelopment. Form B is the most thermodynamically stable crystallinephase under ambient conditions. It can be consistently produced directlyby crystallization. It displays acceptable hygroscopicity and exhibitsgood physical, chemical, and photostability both in solid state and insolution. It also shows acceptable solubility in biorelevant media.Based on the present study and evaluation, Form B is suitable forpharmaceutical development.

Example 6: Crystalline forms

Form A is crystalline but may contain a certain amount of amorphousphase. The DSC curve (FIG. 2 ) of Form A exhibits a dehydrationendotherm at ˜80° C. (peak temperature) which is accompanied by 5.9 wt %weight loss up to ˜150° C. in the TGA curve (FIG. 3 ). The water contentwas found to be 6.4 wt % as determined by a Mettler Toledo DL31 KFTitrator. This data confirms that Form A is a hydrate. Form A ishygroscopic as indicated by water adsorption of ˜5.8 wt % at 80% RHmeasured by dynamic vapor sorption (DVS) (FIG. 4 ). A solid isconsidered moderately hygroscopic when water uptake at 25° C./80% RH isbetween 2-15 wt % based on criteria of European Pharmacopeia. Thehysteresis in the DVS plot suggests that Form A partially converts toForm B during the experiment, although it converts back to Form A whenRH goes back down to zero, therefore no significant change in XRPDpattern was observed post DVS experiment (FIG. 5 ).

Form B is a trihydrate as confirmed by single crystal analysis. Theexperimental XRPD pattern of Form B matches well with the simulated onefrom single crystal data (FIG. 6 ). This was also confirmed by DSC andTGA data (FIG. 7 and FIG. 8 , respectively). DSC data indicates Form Bdehydrates at ˜132° C. (peak temperature) and TGA data displays a 8.73%weight loss up to 160° C. which is in good agreement with thetheoretical value (8.75%) of a trihydrate. Form B is slightlyhygroscopic as evidenced by a weight change of ˜0.3% between 0% and 95%RH (FIG. 9 ), (data collection was programmed from 95% RH-0% RH-95% RHat a step size of 10% RH at 25° C.). XRPD results suggested that no formchange was observed for Form B after the DVS experiment (FIG. 10 ).

Form C was observed upon heating of Form B to 120° C. and cooling downto room temperature (FIG. 11 ). Its DSC and TGA characterization dataare shown in FIG. 12 and FIG. 13 , respectively. The results suggest itis a channel hydrate. Anhydrate Form C material was produced bydehydration of Form B at 114° C. using hot stage XRPD.

Example 7: Single Crystal Structure of EP-1 trihydrate (x is 3)

4 The atomic structure of EP-1 trihydrate (x is 3) is defined by a setof atomic coordinates set forth in Table 18. The terms “structurecoordinates” or “atomic coordinates” refer to Cartesian coordinatesderived from mathematical equations related to the patterns obtained ondiffraction of a monochromatic beam of X-rays by the atoms (scatteringcenters) of a molecule in crystal form. The diffraction data are used tocalculate an electron density map of the repeating unit of the crystal.The electron density maps are then used to establish the positions ofthe individual atoms of the molecule.

TABLE 18 Atomic coordinates and equivalent isotropic atomic displacementparameters (Å²) for EP-1 trihydrate (x is 3). U (eq) is defined as onethird of the trace of the orthogonalized U_(ij) tensor. x/a y/b z/cU(eq) C1 0.3885(4) −0.1399(7) 0.5608(4) 0.0464(16) C2 0.4309(3)−0.0291(6) 0.5967(6) 0.058(2) C3 0.3484(3) −0.2019(6) 0.5980(4)0.0362(14) C4 0.4335(5) 0.0345(7) 0.6574(4) 0.0492(18) C5 0.3528(4)−0.1341(7) 0.6655(4) 0.0504(18) C6 0.3920(4) −0.0215(7) 0.6952(5)0.0506(18) N7 0.4784(3) 0.0490(6) 0.5636(4) 0.0514(16) C8 0.5038(4)0.1426(7) 0.6171(4) 0.0456(17) N9 0.4778(3) 0.1385(7) 0.6711(4)0.0573(17) C10 0.2998(4) −0.3200(7) 0.5720(6) 0.058(2) C11 0.3391(6)−0.4275(9) 0.6342(5) 0.068(2) C12 0.3007(6) −0.3726(11) 0.4964(6)0.080(3) C13 0.2135(4) −0.2985(8) 0.5500(5) 0.058(2) C14 0.5551(4)0.2499(7) 0.6176(4) 0.0435(15) C15 0.5205(3) 0.3815(6) 0.6146(4)0.0361(14) C16 0.5765(4) 0.4863(6) 0.6140(3) 0.0356(14) C17 0.5592(4)0.6261(6) 0.6285(3) 0.0353(14) C18 0.6597(3) 0.4954(6) 0.6853(4)0.0385(15) C19 0.6511(3) 0.6435(6) 0.6811(3) 0.0332(13) N20 0.6775(3)0.7117(5) 0.7560(3) 0.0280(11) C21 0.6458(4) 0.8463(6) 0.7416(4)0.0417(16) C22 0.6768(5) 0.9315(7) 0.7001(5) 0.060(2) C23 0.6586(4)0.9058(6) 0.8188(5) 0.0478(18) C24 0.7659(3) 0.7040(6) 0.8029(3)0.0325(13) C25 0.7952(3) 0.6658(6) 0.8869(3) 0.0334(14) O26 0.7725(2)0.5330(4) 0.8886(2) 0.0343(10) C27 0.8845(4) 0.6622(5) 0.9380(4)0.0377(15) C28 0.8248(3) 0.4807(6) 0.9634(3) 0.0343(13) C29 0.8944(4)0.5746(6) 1.0059(4) 0.0430(16) O30 0.9164(3) 0.7881(4) 0.9641(3)0.0603(17) O31 0.8788(4) 0.6398(6) 1.0616(3) 0.079(2) N32 0.8478(3)0.3506(5) 0.9499(3) 0.0271(11) C33 0.8177(3) 0.2390(7) 0.9620(4)0.0358(14) C34 0.8965(3) 0.3141(5) 0.9183(3) 0.0258(12) N35 0.8432(3)0.1364(5) 0.9431(3) 0.0356(12) C36 0.8935(3) 0.1822(6) 0.9148(3)0.0265(12) C37 0.9385(4) 0.1196(6) 0.8838(4) 0.0366(15) N38 0.9815(3)0.1936(5) 0.8596(3) 0.0339(11) C39 0.9793(3) 0.3228(6) 0.8671(3)0.0320(14) N40 0.9389(3) 0.3914(4) 0.8949(3) 0.0271(11) N41 0.9387(4)−0.0074(5) 0.8747(4) 0.0539(16) O1W 0.5218(4) 0.3015(6) 0.8066(4)0.0682(16) O2W 0.6617(3) 0.4231(4) 0.9663(3) 0.0411(10) O3W 0.5882(2)0.5561(4) 0.8214(2) 0.0371(10)

All publications and patent documents cited herein are incorporatedherein by reference as if each such publication or document wasspecifically and individually indicated to be incorporated herein byreference. Citation of publications and patent documents is not intendedas an admission that any is pertinent prior art, nor does it constituteany admission as to the contents or date of the same. The inventionhaving now been described by way of written description, those of skillin the art will recognize that the invention can be practiced in avariety of embodiments and that the foregoing description and examplesbelow are for purposes of illustration and not limitation of the claimsthat follow.

What is claimed is:
 1. A crystalline form of(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol:

characterized by an X-ray powder diffraction (XRPD) pattern comprisingpeaks at about 16.5, 20.5, and 5.2° 2θ using Cu Kα radiation.
 2. Thecrystalline form of claim 1, characterized by an XRPD pattern comprisingpeaks at about 16.5, 20.5, 5.2, and 14.2° 2θ using Cu Kα radiation. 3.The crystalline form of claim 2, characterized by an XRPD patterncomprising peaks at about 16.5, 20.5, 5.2, 14.2, 18.0, and 10.4° 2θusing Cu Kα radiation.
 4. The crystalline form of claim 1, characterizedby an XRPD pattern comprising at least three peaks selected from thegroup consisting of about 16.5, 20.5, 5.2, 14.2, 18.0, 10.4, 12.3, 10.0,22.7, and 20.9° 2θ using Cu Kα radiation.
 5. The crystalline form ofclaim 1, characterized by an XRPD pattern comprising at least four peaksselected from the group consisting of about 16.5, 20.5, 5.2, 14.2, 18.0,10.4, 12.3, 10.0, 22.7, and 20.9° 2θ using Cu Kα radiation.
 6. Thecrystalline form of claim 1, characterized by an XRPD pattern comprisingat least five peaks selected from the group consisting of about 16.5,20.5, 5.2, 14.2, 18.0, 10.4, 12.3, 10.0, 22.7, and 20.9° 2θ using Cu Kαradiation.
 7. The crystalline form of claim 1, characterized by an XRPDpattern comprising at least six peaks selected from the group consistingof about 16.5, 20.5, 5.2, 14.2, 18.0, 10.4, 12.3, 10.0, 22.7, and 20.9°2θ using Cu Kα radiation.
 8. The crystalline form of claim 1,characterized by an XRPD pattern comprising at least seven peaksselected from the group consisting of about 16.5, 20.5, 5.2, 14.2, 18.0,10.4, 12.3, 10.0, 22.7, and 20.9° 2θ using Cu Kα radiation.
 9. Thecrystalline form of claim 1, characterized by an XRPD pattern comprisingat least eight peaks selected from the group consisting of about 16.5,20.5, 5.2, 14.2, 18.0, 10.4, 12.3, 10.0, 22.7, and 20.9° 2θ using Cu Kαradiation.
 10. The crystalline form of claim 1, characterized by an XRPDpattern comprising at least nine peaks selected from the groupconsisting of about 16.5, 20.5, 5.2, 14.2, 18.0, 10.4, 12.3, 10.0, 22.7,and 20.9° 2θ using Cu Ku radiation.
 11. The crystalline form of claim 1,characterized by an XRPD pattern comprising peaks at about 16.5, 20.5,5.2, 14.2, 18.0, 10.4, 12.3, 10.0, 22.7, and 20.9° 2θ using Cu Kαradiation.
 12. A large-scale process for preparing(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol:

or a salt or hydrate thereof, at about 100 g, about 200 g, about 500 g,about 1 kg, about 2 kg, about 5 kg, about 10 kg, about 20 kg, about 50kg, about 100 kg, about 200 kg, about 500 kg, or about 1000 kgcomprising at least one step selected from the group consisting of: (1)reacting9-((3aR,4R,6R,6aR)-6-(aminomethyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-purin-6-aminewith acetone to yield9-((3aR,4R,6R,6aR)-6-((isopropylamino)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-purin-6-amine;(2) reacting9-((3aR,4R,6R,6aR)-6-((isopropylamino)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-purin-6-aminewith 3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutanoneto yield9-((3aR,4R,6R,6aR)-6-(((3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-purin-6-amine;and (3) deprotecting9-((3aR,4R,6R,6aR)-6-(((3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)-2,2-dimethyltetrahydrofuro[3,4-d][1,3]dioxol-4-yl)-9H-purin-6-amineto afford(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-(((3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol.